Difference between revisions of "Team:TUDelft/Wetlab/Notebook"

Date

05/09/2018

Operator

Lisa Büller

Extraction of 3 serum samples

cfDNA was extracted from 3 serum samples without any spiking of artifical gene doping DNA. For the extraction the QIAamp DNA Blood Mini Kit protocol was used

Qubit dsDNA High Sensitivity DNA Quantification

The 3 extracted samples were quantified using the Qubit dsDNA High Sensitivity DNA Assay Kit.

Sample extraction	cfDNA Concentration (ng/μL)
Serum 1	0.514
Serum 2	0.502
Serum 3	0.530

Date

06/09/2018

Operator

Lisa Büller

Albumin PCR extractions 05/09

GoTaq PCR was done to verify the presence of the albumin gene after extraction of the 3 samples of 05/09/2018. Using the primers creating the small albumin fragment of 153 bp.

Component	Volume (uL)
Samples extr. 1-3 05/09	3
066	0.6
067	0.6
Expected amplicon size (bp)	153

5 tubes were prepared for the PCR, containing the serum extractions of 05/09/2018 as template DNA, a positive control of pure serum and negative control with MiliQ as template. The thermocycler programme was installed to have an extension time of 1 minute and an annealing temperature of 62 °C, 30 cycles. After the programme was finished, samples were kept at 4°C.

The gel showed no results, no bands.

Date

10/09/2018

Operator

Lisa Büller

Extraction of 6 serum samples

cfDNA was extracted from 6 serum samples, of which 3 were spiked with high amounts of Cy5-tagged artificial gene doping EPO DNA as shown in the table. For the extraction the QIAamp DNA Blood Mini Kit protocol was used

Setup of the samples

Sample	Spiked gene doping EPO (ng/ml serum)
Serum 1	0
Serum 2	0
Serum 3	0
Serum 4	60
Serum 5	60
Serum 6	60

Qubit dsDNA High Sensitivity DNA Quantification

The 6 extracted samples were quantified using the Qubit dsDNA High Sensitivity DNA Assay Kit.

Sample	cfDNA Concentration (ng/μL)
Serum 1	0.378
Serum 2	0.380
Serum 3	0.378
Serum 4 - spiked	0.704
Serum 5 - spiked	0.606
Serum 6 - spiked	0.926

10ul of each spiked extraction was loaded on a gel and imaged with the Typhoon. Next to that also 5, 10 and 15 ng of Cy5-labeled EPO where loaded on the gel as reference. Showing that circa 5 ng of Cy5-labeled EPO was present in the 10ul of the extractions. The total extraction volume was 60ul. So circa 5*6=30 ng of Cy5-labeled EPO was present in the final extraction volumes. Showing a roughly 50% extraction efficency, since 60 ng was spiked per sample. Whereas the difference in cfDNA measurement by using the Qubit would suggest an extraction efficiency of circa 37%. But the three spiked samples were too different from eachother in values, probably due to using a too high concentration of sample for spiking.3

Albumin PCR extractions 10/09

GoTaq PCR was done to verify the presence of the albumin gene after extraction of the 6 samples. Using the primers creating the small albumin fragment of 153 bp.

Component	Volume (uL)
Samples extr. 1-6 10/09	3
066	0.6
067	0.6
Expected amplicon size (bp)	153

6 tubes were prepared for the PCR, containing the serum extractions of 10/09/2018 as template DNA. The thermocycler programme was installed to have an extension time of 1 minute and an annealing temperature of 62 °C, 30 cycles. After the programme was finished, samples were kept at 4°C.

The gel showed no results, no bands.

EPO PCR extractions 10/09

GoTaq PCR was done to verify the presence of the EPO gene after extraction of the 3 spiked samples. Using the primers creating the small albumin fragment of 140 bp.

Component	Volume (uL)
Samples extr. 4-6 10/09	3
074	0.6
075	0.6
Expected amplicon size (bp)	140

4 tubes were prepared for the PCR, containing the serum extractions 4-6 of 10/09/2018 as template DNA. As a negative control serum sample 1 was used. The thermocycler programme was installed to have an extension time of 1 minute and an annealing temperature of 62 °C, 30 cycles. After the programme was finished, samples were kept at 4°C.

Date

13/09/2018

Operator

Lisa Büller

Albumin PCR extractions 10/09 - 2^nd round

GoTaq PCR was done to verify the presence of the albumin gene after extraction of the 6 samples. Using the primers creating the small albumin fragment of 153 bp. As a template the PCR product of the previous round of this PCR on the extracted samples were used.

Component	Volume (uL)
Samples extr. PCR 1-6 10/09	3
066	0.6
067	0.6
Expected amplicon size (bp)	153

6 tubes were prepared for the PCR, containing the serum extractions of 10/09/2018 as template DNA. The thermocycler programme was installed to have an extension time of 1 minute and an annealing temperature of 62 °C, 30 cycles. After the programme was finished, samples were kept at 4°C.

The gel showed really faint bands at the expected hight, but also a lot of off target effects and thus different sized bands.

Date

20/09/2018

Operator

Lisa Büller

Extraction of 18 serum samples

cfDNA was extracted from 18 serum samples, of which 15 were spiked piked with artificial gene doping EPO in values coming from the kinetics model as shown in the table. For the extraction the QIAamp DNA Blood Mini Kit protocol was used

Setup of the samples

Sample	Spiked gene doping EPO (ng/ml serum)
Serum 1	0
Serum 2	0
Serum 3	0
Serum 4	0.287 linear EPO
Serum 5	0.287 linear EPO
Serum 6	0.287 linear EPO
Serum 7	0.0319 linear EPO
Serum 8	0.0319 linear EPO
Serum 9	0.0319 linear EPO
Serum 10	0.00127 linear EPO
Serum 11	0.00127 linear EPO
Serum 12	0.00127 linear EPO
Serum 13	0.134 BBa_K2643004
Serum 14	0.134 BBa_K2643004
Serum 15	0.134 BBa_K2643004
Serum 16	0.0053 BBa_K2643004
Serum 17	0.0053 BBa_K2643004
Serum 18	0.0053 BBa_K2643004

Qubit dsDNA High Sensitivity DNA Quantification

The 18 extracted samples were quantified using the Qubit dsDNA High Sensitivity DNA Assay Kit.

Setup of the samples

Sample	cfDNA Concentration (ng/μL)
Serum 1	0.343
Serum 2	0.436
Serum 3	0.356
Serum 4	0.362
Serum 5	0.335
Serum 6	0.392
Serum 7	0.363
Serum 8	0.394
Serum 9	1.18
Serum 10	0.394
Serum 11	0.378
Serum 12	0.432
Serum 13	0.332
Serum 14	0.329
Serum 15	0.366
Serum 16	0.399
Serum 17	0.322
Serum 18	0.322

EPO PCR extractions

GoTaq PCR was done to verify the presence of the spiked EPO gene after extraction of the 18 samples of 20/09/2018.

Component	Volume (uL)
Samples extr. 4-18 20/09	3
074	0.6
075	0.6
Expected amplicon size (bp)	140

19 tubes were prepared for the EPO PCR, containing the serum extractions of 20/09/2018 as template DNA, two positive controls (linear and BB EPO used for spiking), a negative control with MiliQ as template and a non spiked serum extraction as negative control. The thermocycler programme was installed to have an extension time of 40 seconds and an annealing temperature of 65 °C, 30 cycles. After the programme was finished, samples were kept at 4°C.

The PCR gave positive bands for all the spiked samples. But also for the negative control, the unspiked serum. After redoing of this PCR for the non spiked and spiked samples using the two EPO primer combinations 072+073 and 074+075 it became clear that there was a contamination in the non spiked serum samples.

To avoid further contaminations the PCR reagents were tested with PCR on contaminations by using different reagents and also the reagents of the extraction kit were tested. There was no contamination in these reagents.

Albumin PCR extractions 20/09 - 2 rounds

The exact same Albumin PCR steps were followed for the extractions of 20/09 as for the extractions of 10/09 that has positive bands for albumin after two rounds of PCR with the same primers. But, doing this with the samples of 20/09 did not give a positive result. No bands were visible.

Date

05/10/2018

Operator

Lisa Büller

Albumin PCR Serum - 1^st round

GoTaq PCR was done to verify the presence of the albumin gene before extraction of 6 serum samples spiked like the extraction samples of 10/10/2018. First PCR of the nested PCR setup. The serum was spun down at 13.000 xg 4°C fro 30 minutes before it got used as a template. Different volumes of serum were used as template to see the effects of the proteins from the serum on the PCR reaction.

Sample	Template for GoTaq PCR
1 - positive control	3ul extraction 03/10
2 serum	3ul
3 serum	3ul
4 serum	5ul
5 serum	5ul
6 - negative control	3ul MiliQ

Component	Volume (uL)
Serum samples	3-5
091	0.6
092	0.6
Expected amplicon size (bp)	229

6 tubes were prepared for the first round of PCR, containing the serum as template DNA, a positive control extraction from 03/10/2018 and negative control with MiliQ as template. The thermocycler programme was installed to have an extension time of 1 minute and an annealing temperature of 62 °C, 30 cycles. After the programme was finished, samples were kept at 4°C.

Albumin PCR Serum- 2^nd round

GoTaq PCR was done to verify the presence of the albumin gene from the serum samples. Second PCR of the nested PCR setup.

Component	Volume (ul)
Albumin PCR first round serum	3
066	0.6
067	0.6
Expected amplicon size (bp)	153

6 tubes were prepared for the second round of PCR, containing the first round of albumin PCR on the serum as template DNA, a positive control from 03/10/2018 and negative control with MiliQ as template. The same volumes of the new templates were used for the second round as for the first round. The thermocycler programme was installed to have an extension time of 1 minute and an annealing temperature of 62 °C, 30 cycles. After the programme was finished, samples were kept at 4°C.

Date

10/10/2018

Operator

Lisa Büller

Extraction of 6 serum samples - 1^st round

cfDNA was extracted from 6 serum samples, of which some were spiked with artificial gene doping EPO DNA as shown in the table. For the extraction the QIAamp DNA Blood Mini Kit protocol was used

Setup of the samples

Sample	Spiked gene doping EPO (ng/ml serum)
Serum 1	0
Serum 2	0
Serum 3	0.287 linear gene doping EPO
Serum 4	0.287 linear gene doping EPO
Serum 5	1.203 BBa_K2643004
Serum 6	1.203 BBa_K2643004

Qubit dsDNA High Sensitivity DNA Quantification

The 6 extracted samples were quantified using the Qubit dsDNA High Sensitivity DNA Assay Kit.

Sample	cfDNA Concentration (ng/μL)
Serum 1	0.398
Serum 2	0.426
Serum 3	0.386
Serum 4	0.384
Serum 5	0.386
Serum 6	0.386

Date

13/10/2018

Operator

Lisa Büller

Albumin PCR extractions - 1^st round

GoTaq PCR was done to verify the presence of the albumin gene after extraction of the 6 samples of 10/10/2018. First PCR of the nested PCR setup.

Component	Volume (uL)
Samples extr. 1-6 10/10	3
091	0.6
092	0.6
Expected amplicon size (bp)	229

8 tubes were prepared for the first round of PCR, containing the serum extractions of 10/10/2018 as template DNA, a positive control from 03/10/2018 and negative control with MiliQ as template. The thermocycler programme was installed to have an extension time of 1 minute and an annealing temperature of 62 °C, 30 cycles. After the programme was finished, samples were kept at 4°C.

Date

19/07/2018

Operator

Jard Mattens

d-AuNPs Generation

In order to generate dextrin-capped gold nanoparticles, first a gold chloride (HAuCl₄.H₂O) stock solution of 50 mL was prepared by dissolving 393.8 mg of HAuCl₄.H_2O in 50 mL of distilled sterile water. After preparation, this solution was stored at 4 °C covered in aluminium foil.
Secondly, a 10% sterile sodium carbonate (Na₂CO₃) solution was prepared by dissolving 4.985 g Na₂CO₃ in 50 mL MilliQ water, after which the solution was filter sterilized.
Then, 200 mL distilled sterile water was be adjusted to pH 9 by carefully adding 1 M sodium hydroxide (NaOH) drops of 20 uL at a time, while keeping the solution sterile under a flame.
Subsequently, 25 mL of dextrin solution was prepared by dissolving 625 mg of dextrin powder in 25 mL of sterile MilliQ water to obtain a concentration of 25 g/L. This solution was then added to a sterile 250 mL flask.
Then, 5 mL of the gold chloride stock solution was added to the 250 mL flask and the pH was adjusted to 9 with the sterile 10% sodium carbonate solution, while working sterile.
Lastly, the reaction volume was completed to 50 mL by adding 20 mL distilled water (pH = 9).

For the actual generation of the d-AuNPs the flask was then incubated at 50 °C, covered in aluminium foil with continuous shaking for 3 to 8 hours. After 3 to 8 hours shaking, a reddish colour was observed on the solution that is characteristic for the correct size nanoparticle formation. The solution was observed every 30 minutes to check for red color formation.

Transmission Electron Microscope analysis was performed to check the size and size distribution of the d-AuNPs. the suspension of d-AuNPs in distilled water (ratio 1:4) and sonicate it for 5 min. Then, after plasma treatment this solution was be transferred to a copper grid for imaging. The correct size of the d-AuNPs be in the order of 10 nm.

We observed d-AuNPs in a relatively low concentrations. Also, the sizes varied unequally around 100 nm, which is an indication of aggregation as the expected sizes are around 10 nm. Therefore, a new batch had to be made for the actual analysis.

Date

28/08/2018

Operator

Jard Mattens

d-AuNPs Generation

In order to generate dextrin-capped gold nanoparticles, first a gold chloride (HAuCl₄.H₂O) stock solution of 50 mL was prepared by dissolving 393.8 mg of HAuCl₄.H_2O in 50 mL of distilled sterile water. After preparation, this solution was stored at 4 °C covered in aluminium foil.
Secondly, a 10% sterile sodium carbonate (Na₂CO₃) solution was prepared by dissolving 4.985 g Na₂CO₃ in 50 mL MilliQ water, after which the solution was filter sterilized.
Then, 200 mL distilled sterile water was be adjusted to pH 9 by carefully adding 1 M sodium hydroxide (NaOH) drops of 20 uL at a time, while keeping the solution sterile under a flame.
Subsequently, 25 mL of dextrin solution was prepared by dissolving 625 mg of dextrin powder in 25 mL of sterile MilliQ water to obtain a concentration of 25 g/L. This solution was then added to a sterile 250 mL flask.
Then, 5 mL of the gold chloride stock solution was added to the 250 mL flask and the pH was adjusted to 9 with the sterile 10% sodium carbonate solution, while working sterile.
Lastly, the reaction volume was completed to 50 mL by adding 20 mL distilled water (pH = 9).

For the actual generation of the d-AuNPs the flask was then incubated at 50 °C, covered in aluminium foil with continuous shaking for 3 to 8 hours. After 3 to 8 hours shaking, a reddish colour was observed on the solution that is characteristic for the correct size nanoparticle formation. The solution was observed every 30 minutes to check for red color formation.

Transmission Electron Microscope analysis was performed to check the size and size distribution of the d-AuNPs. the suspension of d-AuNPs in distilled water (ratio 1:4) and sonicate it for 5 min. Then, after plasma treatment this solution was be transferred to a copper grid for imaging. The correct size of the d-AuNPs be in the order of 10 nm.

We observed d-AuNPs in relatively low concentrations. However, the sizes were as the expected within a range of 7 nm to 11 nm, which is sufficient for the d-AuNP analysis. In the future however, the protocol should be optimized to generate more uniform batches.

Date

29/08/2018

Operator

Jard Mattens

d-AuNPs Testing: d-AuNPs stability upon salt

The dAuNPs are tested for their stability under different salt concentrations as an initial functionality test. 20 µL of the d-AuNPs solution, 20 µL MilliQ and 20 µL NaCl solutions (range from 0 mM to 1000 mM) were added in one of the 96 wells. The plate was incubated at 21 ºC during 10 minutes.

The aggregation of d-AuNPs, as observed by a colour change from intensely red to purple occured at a final NaCl concentration of 1M*⅓ = 333 mM, but increased when adding more salt.

Date

05/09/2018

Operator

Nicole Bennis

High Fidelity PCR

To amplify the EPO (582 bp) coding sequence, high fidelity PCR was done using the EPO Gblock ordered from IDT as template DNA and 038 and 039 as forward and reverse primers. Two 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

Primer combination(s) and expected fragment(s)

Fw primer	Rv primer	Expected fragment	Expected size (bp)
038	039	EPO	582

The thermocycler programme was installed to have an extension time of 0 seconds and an annealing temperature of 65 °C. After the programme was finished, samples were kept at 4 °C.

Date

10/09/2018

Operator

Nicole Bennis

DNA Gel Electrophoresis

The obtained PCR products with EPO coding sequence were run on a 0.8% agarose gel in TAE buffer. 4 μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

The EPO fragment that should be amplified by the PCR is 582 bp in size. As can be seen on the gel, EPO was successfully amplified.

Date

10/09/2018

Operator

Nicole Bennis

PCR Clean-Up

50 20 μL of each PCR reaction mixture was subjected to PCR Clean-Up, according to the protocol. Elution was done in 30 µL Milli-Q.

Date

10/09/2018

Operator

Nicole Bennis

Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

Isolate	Concentration (ng/uL)	260/280 ratio	260/230 ratio
EPO	56.0	1.90	1.58

Date

14/09/2018

Operator

Nicole Bennis

d-AuNPs Testing: d-AuNPs stability upon salt

The dAuNPs are tested for their stability under different salt concentrations. 20 µL of the d-AuNPs solution, 20 µL MilliQ and 20 µL NaCl solutions (range from 0 mM to 2000 mM) were added in one of the 96 wells on the 92 wells plate. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

The aggregation of d-AuNPs occured at a final NaCl concentration of 1M*⅓ = 333 mM, but increased when adding more salt. At a final concentration of 2M*⅓=666 mM NaCl, the aggregates were the most intensively changed of color. One mistake was made by pipetting the complete d-AuNPs tube into the wells without mixing the tube constantly. The d-AuNPs were sedimentating to the bottom of the tube and this was pipetted into wells E1 and E2. These results should be left out of the data analysis.

From the data, the spectrum of the mixtures with 0 mM, 1000 mM and 2000 mM NaCl were plotted. Due to pipetting errors and mixing errors, the standard deviations at lower salt concentrations were high. Additionally, the from all the samples with different salt concentrations, the ratio 620/520 was calculated and plotted over the salt concentration.

Date

17/09/2018

Operator

Nicole Bennis

d-AuNPs Testing: d-AuNPs stability upon salt

The dAuNPs are tested for their stability under different salt concentrations. 20 µL of the d-AuNPs solution, 20 µL MilliQ and 20 µL NaCl solutions (range from 0 mM to 2000 mM) were added in one of the 96 wells on the 92 wells plate. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

The aggregation of d-AuNPs occured at a final NaCl concentration of 1M*⅓ = 333 mM, but increased when adding more salt. At a final concentration of 2M*⅓=666 mM NaCl, the aggregates were the most intensively changed of color.

From the data, the spectrum of the mixtures with 0 mM, 1000 mM and 2000 mM NaCl were plotted. In comparison with the previous measurements, the error bars are lower. Additionally, the from all the samples with different salt concentrations, the ratio 620/520nm was calculated and plotted over the salt concentration.

Date

17/09/2018

Operator

Nicole Bennis

d-AuNPs Testing: d-AuNPs stability upon salt with ssDNAp

The dAuNPs are tested for their stability under different salt concentrations and the influence of ssDNAp is determined. 20 µL of the d-AuNPs solution, 16 µL MilliQ, 4 µL ssDNAp 1 and 20 µL NaCl solutions (range from 0 mM to 2000 mM) were added in one of the 96 wells on the 96 wells plate. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

The aggregation of d-AuNPs occured at a final NaCl concentration of 1500 mM * ⅓ = 500 mM, but increased when adding more salt. At a final concentration of 2M*⅓=666 mM NaCl, the aggregates were the most intensively changed of color.
From the data, the spectrum of the mixtures with 0 mM, 1000 mM and 2000 mM NaCl were plotted. Additionally, the from all the samples with different salt concentrations, the ratio 620/520 nm was calculated and plotted over the salt concentration.

Date

19/09/2018

Operator

Nicole Bennis

d-AuNPs Testing: d-AuNPs stability upon salt with(out) ssDNAp

The dAuNPs are tested for their stability under different salt concentrations and the influence of ssDNAp is determined. 20 µL of the d-AuNPs solution, 16 µL MilliQ, 4 µL ssDNAp 1 and 20 µL NaCl solutions (range from 0 mM to 2000 mM) were added in one of the 96 wells on the 92 wells plate. As negative controls, 0 L ssDNAp 1 was added. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

The d-AuNPs aggregated when salt was added from a final concentration of 1M*⅓=333 mM to 2M*⅓=666 mM NaCl. When ssDNAp (1 uM) were added, the concentrations at which the d-AuNPs aggregated increased. However, at 2 M NaCl, the d-AuNPs still aggregated although the aggregation was less intense compared to the salt aggregation without ssDNAp’s.

From the data, the spectrum of the mixtures with 0 mM, 1000 mM and 2000 mM NaCl were plotted from both samples: with and without ssDNAp. Additionally, the from all the samples either with or without ssDNAp addition, the ratio 620/520 was calculated and plotted over the salt concentration.

Date

19/09/2018

Operator

Nicole Bennis

d-AuNPs Generation

In order to generate dextrin-capped gold nanoparticles, first a gold chloride (HAuCl₄.H₂O) stock solution of 50 mL was prepared by dissolving 393.8 mg of HAuCl₄.H_2O in 50 mL of distilled sterile water. After preparation, this solution was stored at 4 °C covered in aluminium foil.
Secondly, a 10% sterile sodium carbonate (Na₂CO₃) solution was prepared by dissolving 4.985 g Na₂CO₃ in 50 mL MilliQ water, after which the solution was filter sterilized.
Then, 200 mL distilled sterile water was be adjusted to pH 9 by carefully adding 1 M sodium hydroxide (NaOH) drops of 20 uL at a time, while keeping the solution sterile under a flame.
Subsequently, 25 mL of dextrin solution was prepared by dissolving 625 mg of dextrin powder in 25 mL of sterile MilliQ water to obtain a concentration of 25 g/L. This solution was then added to a sterile 250 mL flask.
Then, 5 mL of the gold chloride stock solution was added to the 250 mL flask and the pH was adjusted to 9 with the sterile 10% sodium carbonate solution, while working sterile.
Lastly, the reaction volume was completed to 50 mL by adding 20 mL distilled water (pH = 9).

For the actual generation of the d-AuNPs the flask was then incubated at 50 °C, covered in aluminium foil with continuous shaking for 3 to 8 hours. After 3 to 8 hours shaking, a reddish colour was observed on the solution that is characteristic for the correct size nanoparticle formation. The solution was observed every 30 minutes to check for red color formation.

Transmission Electron Microscope analysis was performed to check the size and size distribution of the d-AuNPs. the suspension of d-AuNPs in distilled water (ratio 1:4) and sonicate it for 5 min. Then, after plasma treatment this solution was be transferred to a copper grid for imaging. The correct size of the d-AuNPs be in the order of 10 nm.

We observed d-AuNPs in a relatively low concentration. The size distribution is ranging from 8 nm to 13 nm, which is sufficient for d-AuNPs analysis for prescreening.

Date

20/09/2018

Operator

Nicole Bennis

d-AuNPs Testing: d-AuNPs stability upon salt

The newly generated dAuNPs are tested for their stability under different salt concentrations. 20 µL of the d-AuNPs solution, 20 µL MilliQ and 20 µL NaCl solutions (range from 0 mM to 1000 mM) were added in one of the 96 wells on the 92 wells plate. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

With the new batch of d-AuNPs, the salt concentration variation was performed again. The range of salt concentration is adapted from 0 to 1000 mM NaCl added concentration. From the plate view it can be seen that the d-AuNPs are aggregating at a lower salt concentration than the previous (older) batch on 14/09/2018, 17/09/2019 and 19/09/2018.

Date

21/09/2018

Operator

Nicole Bennis

d-AuNPs Testing: ssDNAp interaction with EPO target DNA

The prescreening with d-AuNPs is tested with EPO DNA as target. Prior, the ssDNAp is heated up to let the ssDNAp anneal to EPO target DNA and create the secondary structure. The ssDNAp, EPO target DNA and hybridization buffer were incubated:

• 5 minutes at 95 ºC (denature dsDNA target)
• 1 minute at 57 ºC (anneal ssDNAp to dsDNA target)
• 10 minutes at 21 ºC

20 µL d-AuNPs, 10 µL target dsDNA, 6 µL hybridization buffer, 4 µL ssDNAp 1 and 20 µL of a varying salt concentration (range of 0 mM to 1000 mM) were added to the 96 wells plate. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

The aggregation of d-AuNPs due to increasing salt concentration starts at 500 mM of NaCl. When a ssDNAp is added, the aggregation is extended to a NaCl concentration of 600 mM. However, presence of dsDNA target with a ssDNAp that can specifically bind to the target dsDNA does not influence the aggregation behavior drastically compared to the aggregation when dsDNA target is absent. Presence of only dsDNA target does decrease the stabilization of the d-AuNPs as they already aggregate at a NaCl added of 250 mM.

Date

21/09/2018

Operator

Nicole Bennis

d-AuNPs Testing: ssDNAp interaction with EPO target DNA

The prescreening with d-AuNPs is tested with EPO DNA as target. The ssDNAp concentration is decreased to avoid to much stabilization of d-AuNPs by the ssDNAp. Prior, the ssDNAp is heated up to let the ssDNAp anneal to EPO target DNA and create the secondary structure. The ssDNAp, EPO target DNA and hybridization buffer were incubated:

• 5 minutes at 95 ºC (denature dsDNA target)
• 1 minute at 57 ºC (anneal ssDNAp to dsDNA target)
• 10 minutes at 21 ºC

20 µL d-AuNPs, 10 µL target dsDNA, 6 µL hybridization buffer, 4 µL ssDNAp 1 (1nM) and 20 µL of a varying salt concentration (range of 0 mM to 1000 mM) were added to the 96 wells plate. Again, the primer 051 was used as off-target ssDNAp. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

Date

21/09/2018

Operator

Nicole Bennis

d-AuNPs Testing: ssDNAp interaction with EPO target DNA

The prescreening with d-AuNPs is tested with EPO DNA as target. The ssDNAp concentration is decreased to avoid to much stabilization of d-AuNPs by the ssDNAp. Prior, the ssDNAp is heated up to let the ssDNAp anneal to EPO target DNA and create the secondary structure. The ssDNAp, EPO target DNA and hybridization buffer were incubated:

• 5 minutes at 95 ºC (denature dsDNA target)
• 1 minute at 57 ºC (anneal ssDNAp to dsDNA target)
• 10 minutes at 21 ºC

20 µL d-AuNPs, 10 µL target dsDNA, 6 µL hybridization buffer, 4 µL ssDNAp 1 (100nM) and 20 µL of a varying salt concentration (range of 0 mM to 1000 mM) were added to the 96 wells plate. Again, the primer 051 was used as off-target ssDNAp. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

Date

21/09/2018

Operator

Nicole Bennis

d-AuNPs Testing: ssDNAp interaction with EPO target DNA

The prescreening with d-AuNPs is tested with EPO DNA as target. The ssDNAp concentration is increased to check the stabilization effect on d-AuNPs by the ssDNAp. Prior, the ssDNAp is heated up to let the ssDNAp anneal to EPO target DNA and create the secondary structure. The ssDNAp, EPO target DNA and hybridization buffer were incubated:

• 5 minutes at 95 ºC (denature dsDNA target)
• 1 minute at 57 ºC (anneal ssDNAp to dsDNA target)
• 10 minutes at 21 ºC

20 µL d-AuNPs, 10 µL target dsDNA, 6 µL hybridization buffer, 4 µL ssDNAp 1 (100nM) and 20 µL of a varying salt concentration (range of 0 mM to 1000 mM) were added to the 96 wells plate. Again, the primer 051 was used as off-target ssDNAp. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

Date

02/10/2018

Operator

Nicole Bennis

d-AuNPs Testing: ssDNAp interaction with EPO target DNA

The prescreening with d-AuNPs is tested with EPO DNA as target. To test the sensitivity of the prescreening, the EPO target DNA is lowered to 100 pM. Prior, the ssDNAp is heated up to let the ssDNAp anneal to EPO target DNA and create the secondary structure. The ssDNAp, EPO target DNA and hybridization buffer were incubated:

• 5 minutes at 95 ºC (denature dsDNA target)
• 1 minute at 57 ºC (anneal ssDNAp to dsDNA target)
• 10 minutes at 21 ºC

20 µL d-AuNPs, 10 µL target dsDNA (1 nM and 100 pM), 6 µL hybridization buffer, 4 µL ssDNAp 1 (1 µM) and 20 µL of a varying salt concentration (range of 0 mM to 1000 mM) were added to the 96 wells plate. Again, the primer 051 was used as off-target ssDNAp. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

Date

08/10/2018

Operator

Nicole Bennis

d-AuNPs Testing: ssDNAp interaction with EPO target DNA

The prescreening with d-AuNPs is tested with EPO DNA as target. To test the versatility of the prescreening, multiple variants of the ssDNAp were designed to target the EPO DNA. The ssDNAp 2 targets the EPO coding sequence. Prior, the ssDNAp is heated up to let the ssDNAp anneal to EPO target DNA and create the secondary structure. The ssDNAp 2 was used in this experiment to target the EPO DNA. The ssDNAp 2, EPO target DNA and hybridization buffer were incubated:

• 5 minutes at 95 ºC (denature dsDNA target)
• 1 minute at 57 ºC (anneal ssDNAp to dsDNA target)
• 10 minutes at 21 ºC

20 µL d-AuNPs, 10 µL target dsDNA (1 µM), 6 µL hybridization buffer, 4 µL ssDNAp 2 (1 µM) and 20 µL of a varying salt concentration (range of 0 mM to 1000 mM) were added to the 96 wells plate. Again, the primer 051 was used as off-target ssDNAp. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

Date

08/10/2018

Operator

Nicole Bennis

d-AuNPs Testing: ssDNAp interaction with EPO target DNA

The prescreening with d-AuNPs is tested with EPO DNA as target. To test the versatility of the prescreening, multiple variants of the ssDNAp were designed to target the EPO DNA. The ssDNAp 3 targets the EPO coding sequence. Prior, the ssDNAp is heated up to let the ssDNAp anneal to EPO target DNA and create the secondary structure. The ssDNAp 3 was used in this experiment to target the EPO DNA. The ssDNAp 3, EPO target DNA and hybridization buffer were incubated:

• 5 minutes at 95 ºC (denature dsDNA target)
• 1 minute at 57 ºC (anneal ssDNAp to dsDNA target)
• 10 minutes at 21 ºC

20 µL d-AuNPs, 10 µL target dsDNA (1 µM), 6 µL hybridization buffer, 4 µL ssDNAp 3 (1 µM) and 20 µL of a varying salt concentration (range of 0 mM to 1000 mM) were added to the 96 wells plate. Again, the primer 051 was used as off-target ssDNAp. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

Date

08/10/2018

Operator

Nicole Bennis

d-AuNPs Testing: ssDNAp interaction with EPO target DNA

The prescreening with d-AuNPs is tested with EPO DNA as target. To test the versatility of the prescreening, multiple variants of the ssDNAp were designed to target the EPO DNA. The ssDNAp 4 targets the EPO coding sequence. Prior, the ssDNAp is heated up to let the ssDNAp anneal to EPO target DNA and create the secondary structure. The ssDNAp 4 was used in this experiment to target the EPO DNA. The ssDNAp 4, EPO target DNA and hybridization buffer were incubated:

• 5 minutes at 95 ºC (denature dsDNA target)
• 1 minute at 57 ºC (anneal ssDNAp to dsDNA target)
• 10 minutes at 21 ºC

20 µL d-AuNPs, 10 µL target dsDNA (1 µM), 6 µL hybridization buffer, 4 µL ssDNAp 4 (1 µM) and 20 µL of a varying salt concentration (range of 0 mM to 1000 mM) were added to the 96 wells plate. Again, the primer 051 was used as off-target ssDNAp. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

Date

08/10/2018

Operator

Nicole Bennis

d-AuNPs Testing: ssDNAp interaction with EPO target DNA

The prescreening with d-AuNPs is tested with EPO DNA as target. To test the versatility of the prescreening, multiple variants of the ssDNAp were designed to target the EPO DNA. The ssDNAp 5 targets the EPO coding sequence. Prior, the ssDNAp is heated up to let the ssDNAp anneal to EPO target DNA and create the secondary structure. The ssDNAp 5 was used in this experiment to target the EPO DNA. The ssDNAp 5, EPO target DNA and hybridization buffer were incubated:

• 5 minutes at 95 ºC (denature dsDNA target)
• 1 minute at 57 ºC (anneal ssDNAp to dsDNA target)
• 10 minutes at 21 ºC

20 µL d-AuNPs, 10 µL target dsDNA (1 µM), 6 µL hybridization buffer, 4 µL ssDNAp 5 (1 µM) and 20 µL of a varying salt concentration (range of 0 mM to 1000 mM) were added to the 96 wells plate. Again, the primer 051 was used as off-target ssDNAp. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

Date

09/10/2018

Operator

Nicole Bennis

d-AuNPs Testing: ssDNAp interaction with EPO target DNA

The prescreening with d-AuNPs is tested with EPO DNA as target. To test the effect of background DNA on the ability to screen for target DNA, linear DNA was added. The ssDNAp 1 was used in this experiment to target the EPO DNA while background DNA is present. The ssDNAp 1, EPO target DNA, background DNA and hybridization buffer were incubated:

• 5 minutes at 95 ºC (denature dsDNA target)
• 1 minute at 57 ºC (anneal ssDNAp to dsDNA target)
• 10 minutes at 21 ºC

20 µL d-AuNPs, 10 µL target dsDNA (1 µM), 6 µL hybridization buffer, 4 µL ssDNAp 1 (1 µM) and 20 µL of a varying salt concentration (range of 0 mM to 1000 mM) were added to the 96 wells plate. Again, the primer 051 was used as off-target ssDNAp. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

Date

09/10/2018

Operator

Nicole Bennis

d-AuNPs Testing: ssDNAp interaction with EPO target DNA

The prescreening with d-AuNPs is tested with EPO DNA as target. To test the effect of background DNA on the ability to screen for target DNA, linear DNA was added. The ssDNAp 1 was used in this experiment to target the EPO DNA while background DNA is present. The ssDNAp 1, EPO target DNA, background DNA and hybridization buffer were incubated:

• 5 minutes at 95 ºC (denature dsDNA target)
• 1 minute at 57 ºC (anneal ssDNAp to dsDNA target)
• 10 minutes at 21 ºC

20 µL d-AuNPs, 10 µL target dsDNA (1 µM), 6 µL hybridization buffer, 4 µL ssDNAp 2 (1 µM) and 20 µL of a varying salt concentration (range of 0 mM to 1000 mM) were added to the 96 wells plate. Again, the primer 051 was used as off-target ssDNAp. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

In order to check the versatility of the prescreening, the effect of background DNA present in the sample, the d-AuNPs test is also performed with ssDNAp 3, 4 and 5.

Date

30/5/2018

Operator

Kavish Kohabir

Plasmid Isolation

An overnight starter culture of E. coli DH5α harboring plasmid pTXB1-Tn5 was subjected to plasmid isolation following the Plasmid Isolation Protocol. Final elution was done with 30 μL of pre-warmed MilliQ.

Date

30/5/2018

Operator

Kavish Kohabir

Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

Isolate	Concentration (ng/uL)	260/280 ratio	260/230 ratio
pTXB1-Tn5	36.3	1.92	2.06
pTXB1-Tn5	40.4	1.88	2.17
pTXB1-Tn5	47	1.82	1.75

Date

1/6/2018

Operator

Kavish Kohabir

Primer working stock preparation

The following primers were resuspended in sterile Milli-Q water, in order to make 100µM stock solutions.

Primer name	Amount (ng)	Volume (uL)
001	290	277
002	380	304
003	280	377
004	280	344
005	4nmol	40*
006	260	255
007	290	284
008	4nmol	40*
009	250	290
010	270	320
011	220	308
012	200	301
013	220	357
014	180	301
015	200	334
016	200	330
017	190	350
018	170	348
019	290	345
020	220	365
021	240	343
022	230	334
023	200	319
024	230	336
025	220	339
026	160	355
027	150	301
028	170	343

The primers in these 100 µM stocks were heated to 60 °C for 20 minutes and centrifuged at maximum speed (~17,000 x g) for 2 minutes. 10µM primer working stock solutions were prepared by making 10x dilution aliquots with sterile Milli-Q, each in a total volume of 100µl.

Date

04/06/2018

Operator

Nicole Bennis

High Fidelity PCR

To amplify Linker-Tn5 (1508 bp), Tn5 (1452 bp) and Tn5-Linker (1505 bp) coding sequence, high fidelity PCR was done using pTXB1-Tn5 as template DNA and FP005, FP006, FP007 and FP008 as forward and reverse primers. Two 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

Primer combination(s) and expected fragment(s)

Sample	Fw primer	Rv primer	Expected fragment	Expected size (bp)
1	005	006	Linker-Tn5	1508
2	007	006	Tn5	1452
3	007	008	Tn5-Linker	1505

The thermocycler programme was installed to have an extension time of 1 min 30 seconds and an annealing temperature of 55 °C for one of the tubes and 60 °C for the second tubes. After the programme was finished, samples were kept at 4 °C.

Date

04-06-2018

Operator

Nicole Bennis

DNA Gel Electrophoresis

The obtained PCR products Linker-Tn5, Tn5, Tn5-Linker were run on a 0.8% agarose gel in TAE buffer. 4 μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

The fragments that should be amplified by the PCR are respectively 1508, 1452 and 1505 bp in size. As can be seen on the gel, only the Tn5 amplification was successfully amplified. The Linker-Tn5 and Tn5-Linker fragments were not successfully amplified.

Date

05/06/2018

Operator

Nicole Bennis

PCR clean-up

Two times 50 μL of PCR product Tn5 was subjected to PCR Clean-Up, according to the DNA clean up protocol. Elution was done in 30 µL prewarmed Milli-Q.

Date

05-06-2018

Operator

Nicole Bennis

Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

Isolate	Concentration (ng/uL)	260/280 ratio	260/230 ratio
Tn5 (annealed at 55 °C)	59.6	1.88	1.65
Tn5 (annealed at 60 °C)	47.4	1.82	0.59

Date

05/06/2018

Operator

Nicole Bennis

Liquid Starter Culture Preparation

E.coli strain harbouring the plasmid pTXB1-Tn5 was used for inoculation of 10 mL liquid starter culture (Luria Broth, complemented with ampicillin) following the Liquid Starter Culture Protocol.

Date

06/06/2018

Operator

Nicole Bennis

Restriction

Restriction cloning was done for assembling Tn5 into pACYC-Duet. The following table shows an overview of what fragment(s) were cut with which restriction enzyme(s).

Component	Volume (uL)
10x CutSmart buffer	2.5
Fragment (pACYC-Duet)	10 (1000 ng)
Enzyme (EcoRI)	1
Enzyme (KpnI)	1
MilliQ	10.5

Component	Volume (uL)
10x CutSmart buffer	2.5
Fragment (Tn5)	20.5 (1000 ng)
Enzyme (EcoRI)	1
Enzyme (KpnI)	1
MilliQ	0

The samples were incubated for 2 hours at 37 °C.

Date

08/06/2018

Operator

Nicole Bennis

PCR Clean up

25 μL of restriction mixture with Tn5 or pACYCDuet-1 was subjected to DNA Clean-Up, according to the protocol. Elution was done in 30 µL prewarmed Milli-Q.

Date

08/06/2018

Operator

Nicole Bennis

Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

Isolate	Concentration (ng/uL)	260/280 ratio	260/230 ratio
Tn5 55 °C (EcoRI, KpnI restricted)	8.8	1.97	0.79
Tn5 60 °C (EcoRI, KpnI restricted)	6.8	1.68	0.52
Duet-1 (EcoRI, KpnI restricted)	5.6	2.05	0.40

Date

08/06/2018

Operator

Nicole Bennis

Ligation

In order to assemble cut fragments Tn5 55 °C/60 °C and pACYCDuet-1, ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

DNA	Size (bp)	Cut with enzymes
Vector pACYCDuet-1	3768	EcoRI, KnpI
Fragment Tn5	1442	EcoRI, KpnI

A 20 μL ligation reaction mixture was prepared with the following composition:

Component	Volume (uL)
Ligase Buffer (10x)	2
T4 DNA Ligase	1
DNA vector (~50 ng)	9
DNA fragment (~60 ng)	8
MilliQ	0

The samples were incubated for 72 hours (over the weekend) at 4°C for ligation.

Date

08/06/2018

Operator

Nicole Bennis

High Fidelity PCR

To amplify Linker-Tn5 (1508 bp), Tn5 (1452 bp) and Tn5-Linker (1505 bp) coding sequence, high fidelity PCR was done using pTXB1-Tn5 as template DNA and FP005, FP006, FP007 and FP008 as forward and reverse primers. As the PCR reaction was not successful before, different conditions were tested to optimize the PCR. Multiple 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.
Primer combination(s) and expected fragment(s)

Sample	Fw primer	Rv primer	Expected fragment	Expected size (bp)
1	005	006	Linker-Tn5	1508
2	007	006	Tn5	1452
3	007	008	Tn5-Linker	1505

The sets of conditions tested were:

• Touch Down PCR with HF buffer
• Touch Down PCR with GC buffer
• 2 step PCR with HF buffer
• 2 step PCR with GC buffer

Touch Down PCR program

Step	Time (s)	Temperature (°C)
Initial denaturation	30	98
Denaturation	10	98
Annealing	15	60 down to 50
Extension	1 min 30 sec	72
Final extension	300	72
Hold	∞	4

2 Step PCR program

Step	Time (s)	Temperature (°C)
Initial denaturation	30	98
Denaturation	10	98
Annealing	15	72
Extension	1 min 30 sec	72
Final extension	300	72
Hold	∞	4

After the programme was finished, samples were kept at 4 °C.

Date

08/06/2018

Operator

Nicole Bennis

DNA Gel Electrophoresis

The obtained PCR products Linker-Tn5, Tn5, Tn5-Linker were run on a 0.8% agarose gel in TAE buffer. 4 μL DNA was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

The fragments that should be amplified by the PCR are respectively 1508, 1452 and 1505 bp in size. As can be seen on the gel, only the Linker-Tn5 amplification was successfully amplified in the 2 Step PCR program with GC buffer. The Linker-Tn5, Tn5 and Tn5-Linker fragments were not successfully amplified when a touch down PCR program was performed.

Date

08/06/2018

Operator

Nicole Bennis

High Fidelity PCR

To amplify Linker-Tn5 (1508 bp), Tn5 (1452 bp) and Tn5-Linker (1505 bp) coding sequence, high fidelity PCR was done using the amplified Tn5 (in previous PCR reactions) as template DNA and FP005, FP006, FP007 and FP008 as forward and reverse primers. Two times eight 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

Primer combination(s) and expected fragment(s)

Sample	Fw primer	Rv primer	Expected fragment	Expected size (bp)
1	005	006	Linker-Tn5	1508
2	007	008	Tn5-Linker	1505

To test the effect of DMSO on the PCR performance, one extra tube per amplicon was made that was supplemented with 1.5 μL DMSO (3% final) and 34 μL MilliQ. Additionally, instead of the amplified Tn5 PCR product as template, the original pTXB1-Tn5 plasmid was used as template. Finally, a negative control sample was taken into account that contained no primers and 37.5 μL MilliQ instead.
The thermocycler programme was installed to have an extension time of 1 min 30 seconds and an annealing temperature of 60 °C. In order to check the difference between two PCR machines (older one and new one), the tubes were divided over the two thermocyclers. After the programme was finished, samples were kept at 4 °C.

Date

08/06/2018

Operator

Nicole Bennis

DNA Gel Electrophoresis

The obtained PCR products Linker-Tn5 and Tn5-Linker were run on a 0.8% agarose gel in TAE buffer. 4 μL DNA was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

As can be seen, the PCR amplification with primers 005 and 006 resulting in Linker-Tn5 were successful. The primers 007 and 008 did not result in any PCR amplicons. Also, the HF buffer supplemented with 3% DMSO resulted in the required amplicon. Remarkably, the old PCR machine did result in thicker and brighter bands DNA. This might be due to the differences in speed when changing temperature.

Date

08/06/2018

Operator

Nicole Bennis

Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

Isolate	Concentration (ng/uL)	260/280 ratio	260/230 ratio
Linker-Tn5, 7 tubes (new machine)	117.9	1.82	1.35
Linker-Tn5, 1 tube (old machine)	67.7	1.82	1.17

Date

08-06-2018

Operator

Nicole Bennis

High Fidelity PCR

To amplify Linker-Tn5 (1508 bp), Tn5 (1452 bp) and Tn5-Linker (1505 bp) coding sequence, high fidelity PCR was done using pTXB1-Tn5 as template DNA and FP005, FP006, FP007 and FP008 as forward and reverse primers. Two times three 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

Primer combination(s) and expected fragment(s)

Sample	Fw primer	Rv primer	Expected fragment	Expected size (bp)
1	005	006	Linker-Tn5	1508
2	007	006	Tn5	1452
3	007	008	Tn5-Linker	1505

The thermocycler programme was installed to have an extension time of 2 min 30 seconds (1 minute longer than PCR reactions before) and an annealing temperature of 60 °C. Again, in order to check the difference between two PCR machines (older one and new one), the tubes were divided over the two thermocyclers. After the programme was finished, samples were kept at 4 °C.

Date

08-06-2018

Operator

Nicole Bennis

DNA Gel Electrophoresis

All the PCR amplicons were run on a 0.8% agarose gel in TAE buffer. 4 μL DNA was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

As can be seen, the PCR amplification with primers 005, 006, 007 and 008 resulting in Linker-Tn5, Tn5, Tn5-Linker (respective sizes: 1508, 1452 and 1505 bp) were successful. In contradiction with the previous results, the new PCR machine did result in thicker and brighter bands DNA and is therefore considered more efficient for this PCR reaction. From this experiment it can be concluded that the standard PCR conditions work sufficient for this purpose.

Date

11/06/2018

Operator

Nicole Bennis

Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

Strain	Plasmid and size (bp)	Cloning method	Volume added (μl)	Medium on which was plated
E.coli DH5α	pACYC1Duet-1_Tn5 (5210 bp)	Restriction/Ligation	20	LB Cam
E.coli DH5α	pACYCDuet-1_Tn5(5210 bp)	Restriction/Ligation	20	LB Cam
E.coli DH5α	pTXB1-Tn5	-	2	LB Amp
E.coli DH5α	pACYCDuet-1 (pos.control)	-	1	LB Cam
E.coli DH5α	none (neg. control)	Restriction/Ligation	0	LB Cam

200 μl LB medium was added as recovery medium.

Date

12/06/2018

Operator

Nicole Bennis

Colony PCR

Colony PCR was done to verify propagation of pACYC1Duet-1_Tn5 after transformation from 11-06-2018. This plasmid contains the Tn5 insert, which can be checked with primers 029 and 030. As negative control, the colonies containing pACYCDuet-1 were checked according to the same protocol.

Template DNA	pACYCDuet-1_Tn5
Fw primer	029
Rv primer	030
expected amplicon size (bp)	1686 bp

Six tubes were prepared for colony PCR, containing pACYCDuet-1_Tn5 as template DNA. Additionally, one tube with pACYCDuet-1 was taken along as positive control. The thermocycler programme was installed to have an extension time of 2 min and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

Date

13/06/2018

Operator

Nicole Bennis

DNA Gel Electrophoresis

The PCR amplicons of the colony PCR performed on 12-06-2018 were run on a 0.8% agarose gel in TAE buffer. 4μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

Unfortunately, the PCR reaction did not result in the required PCR amplicons. However, the positive control colony (pACYCDuet-1) did also not results in any PCR amplicon. Therefore, it is likely that the PCR reaction itself was not successful and needs to be repeated.

Date

13-06-2018

Operator

Nicole Bennis

PCR Clean-Up

The PCR amplicons made on 08-06-2018 were subjected to PCR Clean-Up, according to the protocol. Elution was done in 30 µL Milli-Q.

Date

13-06-2018

Operator

Nicole Bennis

Nanodrop DNA Quantification

The PCR amplicons were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

Isolate	Concentration (ng/uL)	260/280 ratio	260/230 ratio
Linker-Tn5	494.1	1.88	1.95
Tn5	463.1	1.86	1.90
Tn5-Linker	398.3	1.88	1.87

Date

14/06/2018

Operator

Nicole Bennis

Liquid Starter Culture Preparation

The six colonies that have grown on LB Cam after transformation on 11-06-2018 and probably harbour our desired plasmid pACYCDuet-1_Tn5 were used for inoculation of 50 mL liquid starter culture (Luria Broth, complemented with Cam) following the Liquid Starter Culture Protocol. Additionally, as positive control one colony of pACYCDuet-1 that has also grown was inoculated in the same way.

Date

15/06/2018

Operator

Nicole Bennis

Restriction

Restriction cloning was done for assembling Tn5, Tn5-Linker and Linker-Tn5 separately into pACYCDuet-1. The following table shows an overview of what fragment(s) was/were cut with what restriction enzyme(s).

Component	Volume (uL)	Component	Volume (uL)	Component	Volume (uL)
10x CutSmart buffer	5	10x CutSmart buffer	5	10x CutSmart buffer	5
Fragment (Tn5)	4.5 (1000 ng)	Fragment (Linker-Tn5)	4 (1000 ng)	Fragment (Tn5-Linker)	5 (1000 ng)
Enzyme (EcoRI)	1	Enzyme (FseI)	1	Enzyme (EcoRI)	1
Enzyme (KpnI)	1	Enzyme (KpnI)	1	Enzyme (NotI)	1
MilliQ	38.5	MilliQ	39	MilliQ	38

The samples were incubated for 1 hour at 37 °C, and followed by 20 minute at 65 °C heat inactivation of the restriction enzyme(s).

Date

15/06/2018

Operator

Timothy Páez

Restriction

Restriction cloning was done for assembling Tn5-lin and lin-Tn5 into pACYCduet-1. The following table shows an overview of what fragments were cut with what restriction enzymes.

Component	Volume (uL)
10x CutSmart buffer	2
Fragment (Tn5-lin)	9
Enzyme (ECORI HF)	1
Enzyme (NotI HF)	1
MilliQ	7

Component	Volume (uL)
10x CutSmart buffer	2
Vector (pACYC)	16
Enzyme (EcoRI HF)	1
Enzyme (NotI HF)	1
MilliQ	0

The samples were incubated for 1 hour at 37 °C.

Date

15/06/2018

Operator

Timothy Páez

PCR clean-up

20 μL of restriction product were subjected to PCR Clean-Up, according to the protocol. Elution was done in 20 µL Milli-Q.

Products were quantified with Nanodrop, yielding concentration of:
- Tn5-lin: 30.4 ng/µL
- pACYC: 8.5 ng/µL

Date

15/06/2018

Operator

Timothy Páez

Ligation

In order to assemble cut fragments Tn5-lin into pACYCduet-1, ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

DNA	Size (bp)	Cut with enzymes
pACYCDuet	1.5 kbp	EcoRI and NotI
Tn5-lin	1.5 kbp	EcoRI and NotI

The ligation reaction mixture was prepared with the following composition

Component	Volume (uL)
Ligase Buffer (10x)	2 µL
T4 DNA Ligase	1 µL
pACYC (~100 ng)	13.3 µL
Tn5 (300 ng)	3.7 µL
MilliQ	0 µL

The samples were incubated overnight at 4 ºC for ligation.

Date

16/06/2018

Operator

Timothy Páez

Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

Strain	Plasmid & size (bp)	Cloning method	Volume added (uL)	Medium on which was plated
E.coliDH5α	pACYC Tn5 (3000 bp)	Restriction Ligation	10 µL	LB with Chloroamphenicol

400 µL LB medium was added as recovery medium.

Date

17/06/2018

Operator

Timothy Páez

Transformation results from 16/06/2018

No colonies grew on LB plates with chloroamphenicol. Repeat protocol with higher DNA concentrations starting from restriction.

Date

18-06-2018

Operator

Nicole Bennis

Plasmid Isolation

Overnight starter cultures of Tn5 (two times) and pACYCDuet-1 (two times) were subjected to plasmid isolation following the Plasmid Isolation Protocol. Final elution was done with 30 μL of pre-warmed MilliQ.

Date

18-06-2018

Operator

Nicole Bennis

Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

Isolate	Concentration (ng/uL)	260/280 ratio	260/230 ratio
Tn5 (1)	119.1	2.02	2.08
Tn5 (2)	127.3	2.00	2.32
pACYCDuet-1 (1)	47.4	1.96	2.03
pACYCDuet-1 (2)	238.9	2.12	2.44

Date

18-06-2018

Operator

Nicole Bennis

Restriction

Restriction cloning was done for assembling Tn5, Tn5-Linker and Linker-Tn5 separately into pACYCDuet-1. The following table shows an overview of what fragment(s) was/were cut with what restriction enzyme(s).

Component	Volume (uL)	Component	Volume (uL)	Component	Volume (uL)	Component	Volume (uL)
10x CutSmart buffer	5	10x CutSmart buffer	5	10x CutSmart buffer	5	10x CutSmart buffer	5
Fragment (Tn5)	4.5 (2000 ng)	Fragment (Linker-Tn5)	4 (2000 ng)	Fragment (Tn5-Linker)	5 (2000 ng)	Fragment (pACYCDuet-1)	5 (2000 ng)
Enzyme (EcoRI)	1	Enzyme (FseI)	1	Enzyme (EcoRI)	1	Enzyme (EcoRI)	1
Enzyme (KpnI)	1	Enzyme (KpnI)	1	Enzyme (NotI)	1	Enzyme (NotI)	1
MilliQ	38.5	MilliQ	39	MilliQ	38	MilliQ	38

The samples were incubated for 1 hour at 37 °C, and followed by 20 minute at 65 °C heat inactivation of the restriction enzyme(s).

Date

18/06/2018

Operator

Nicole Bennis

PCR Clean-Up

The restriction reactions Tn5, Tn5-Linker, Linker-Tn5 and pACYCDuet-1 were subjected to PCR Clean-Up, according to the protocol. Elution was done in 30 µL Milli-Q.

Date

18-06-2018

Operator

Nicole Bennis

Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

Strain	Plasmid and size (bp)	Cloning method	Volume added (μl)	Medium on which was plated
E.coli DH5α	pACYCDuet-1_Tn5 (5210 bp)	Restriction/Ligation	5	LB Cam
E.coli DH5α	pACYCDuet-1_Linker-Tn5 (5477 bp)	Restriction/Ligation	5	LB Cam
E.coli DH5α	pACYCDuet-1 restricted (neg. control)	Restriction	5	LB Cam
E.coli DH5α	pACYCDuet-1 (pos. control)	-	0.5	LB Cam

200 μl LB medium was added as recovery medium.

Date

08-06-2018

Operator

Nicole Bennis

Liquid Starter Culture Preparation

The colonies that have grown on the plates of the transformation performed on 11-06-2018 were used for inoculation of 5 mL liquid starter culture (Luria Broth, complemented with chloramphenicol) following the Liquid Starter Culture Protocol. This was done to be able to test the colonies for correct insert.

Date

19/06/2018

Operator

Nicole Bennis

High Fidelity PCR

To amplify Linker-Tn5 (1508 bp), Tn5 (1452 bp) and Tn5-Linker (1505 bp) coding sequence, high fidelity PCR was done using pTXB1-Tn5 as template DNA and FP005, FP006, FP007 and FP008 as forward and reverse primers. Two times three 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

Primer combination(s) and expected fragment(s)

Sample	Fw primer	Rv primer	Expected fragment	Expected size (bp)
1	005	006	Linker-Tn5	1508
2	007	006	Tn5	1452
3	007	008	Tn5-Linker	1505

The thermocycler programme was installed to have an extension time of 2 min 30 seconds (1 minute longer than PCR reactions before) and an annealing temperature of 60 °C. Again, in order to check the difference between two PCR machines (older one and new one), the tubes were divided over the two thermocyclers. After the programme was finished, samples were kept at 4 °C.

Date

19/06/2018

Operator

Nicole Bennis

Plasmid Isolation

Overnight starter cultures of the colonies from the transformation performed on 11-06-2018 were subjected to plasmid isolation following the Plasmid Isolation Protocol. Final elution was done with 20 μL of pre-warmed MilliQ.

Date

19/06/2018

Operator

Nicole Bennis

Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

Isolate	Concentration (ng/uL)	260/280 ratio	260/230 ratio
Colony 1: pACYCDuet-1_Tn5	19.7	1.75	0.86
Colony 2: pACYCDuet-1_Tn5	38.6	1.66	0.79
Colony 3: pACYCDuet-1_Tn5	93.4	1.56	0.62
Colony 4: pACYCDuet-1_Tn5	34.6	1.75	0.98
Colony 5: pACYCDuet-1_Tn5	57.4	1.62	0.74
Colony 6: pACYCDuet-1_Tn5	39.8	1.73	0.99
Colony 7: pACYCDuet-1_Tn5	32.5	1.77	1.10
Colony pACYCDuet-1	54.3	1.73	1.05

Date

19/06/2018

Operator

Nicole Bennis

Colony PCR

Colony PCR was done to verify propagation of pACYCDuet-1_Tn5 after transformation from 11-06-2018. This plasmid contains Tn5, which can be checked with primers 006 and 007. Additionally, the primers binding at the T7 promoter and termination were used to check the Tn5 insert.

Template DNA	pACYCDuet-1_Tn5	pACYCDuet-1_Tn5
Fw primer	007	029
Rv primer	006	030
expected amplicon size (bp)	1452 bp	1576 bp

Eight tubes were prepared for colony PCR, containing pACYCDuet-1-Tn5 as template DNA. Additionally, MgCl₂ was added to increase the efficiency of the PCR reaction. The thermocycler programme was installed to have an extension time of 2 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

Date

19/06/2018

Operator

Nicole Bennis

DNA Gel Electrophoresis

The colony PCR’s of the transformation of 11-06-2018 were run on a 0.8% agarose gel in TAE buffer. 5 μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

Colonies 1,2,3,4,6 and 7 show the presence of Tn5 and T7p-Tn5-T7t, indicating that these colonies were positive for Tn5 integration.

Date

19/06/2018

Operator

Nicole Bennis

High Fidelity PCR

To amplify Linker-Tn5 (1508 bp), Tn5 (1452 bp) and Tn5-Linker (1505 bp) coding sequence, high fidelity PCR was done using pTXB1-Tn5 as template DNA and FP005, FP006, FP007 and FP008 as forward and reverse primers. Multiple 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.
Primer combination(s) and expected fragment(s)

Sample	Fw primer	Rv primer	Expected fragment	Expected size (bp)
1	005	006	Linker-Tn5	1508
2	007	006	Tn5	1452
3	007	008	Tn5-Linker	1505

The thermocycler programme was installed to have an extension time of 2 min 30 seconds (1 minute longer than PCR reactions before) and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

Date

19/06/2018

Operator

Nicole Bennis

Plasmid Isolation

Overnight starter cultures of the strain with pACYCDuet-1 were subjected to plasmid isolation following the Plasmid Isolation Protocol. Final elution was done with 30 μL of pre-warmed MilliQ.

Date

19/06/2018

Operator

Nicole Bennis

Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

Isolate	Concentration (ng/uL)	260/280 ratio	260/230 ratio
pACYCDuet-1 (1)	15.5	1.85	1.36
pACYCDuet-1 (2)	14.0	1.60	0.86

Date

19-06-2018

Operator

Nicole Bennis

Liquid Starter Culture Preparation

Colonies 1,2,3,4,6 and 7 harbouring the pACYCDuet-1_Tn5 plasmid and strains containing the plasmids pTXB1-Tn5 and pACYCDuet-1 were used for inoculation of 50 mL liquid starter culture (Luria Broth, complemented with chloramphenicol) following the Liquid Starter Culture Protocol.

Date

20/06/2018

Operator

Nicole Bennis

Plasmid Isolation

Overnight starter cultures of pACYCDuet-1 and pTXB1-Tn5 were subjected to plasmid isolation following the Plasmid Isolation Protocol. Final elution was done with 30 μL of pre-warmed MilliQ.

Date

20/06/2018

Operator

Nicole Bennis

Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

Isolate	Concentration (ng/uL)	260/280 ratio	260/230 ratio
pACYCDuet-1 (1)	120.9	1.67	0.85
pACYCDuet-1 (2)	108.1	1.70	1.03
pACYCDuet-1 (3)	56.8	1.86	1.52
pACYCDuet-1 (4)	113.8	1.68	0.91
pTXB1-Tn5 (1)	90.6	1.81	1.45
pTXB1-Tn5 (2)	96.6	1.82	1.65
pTXB1-Tn5 (3)	106.4	1.79	1.36
pTXB1-Tn5 (4)	121.7	1.73	1.18

Date

20/06/2018

Operator

Nicole Bennis

PCR Clean-Up

Two sets of 50 μL of PCR product of Tn5, Linker-Tn5 and Tn5-linker performed on and 19-06-2018 was subjected to PCR Clean-Up, according to the protocol. Elution was done in 30 µL prewarmed Milli-Q.

Date

20/06/2018

Operator

Nicole Bennis

Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

Isolate	Concentration (ng/uL)	260/280 ratio	260/230 ratio
Tn5-Linker (1)	190.3	1.90	1.38
Tn5-linker (2)	206.0	1.90	1.98
Tn5 (1)	258.4	1.88	2.04
Tn5 (2)	260.7	1.88	2.06
Linker-Tn5 (1)	199.7	1.88	1.86
Linker-Tn5 (2)	234.6	1.89	1.99

Date

20/06/2018

Operator

Nicole Bennis

Restriction

Restriction cloning was done for assembling Tn5, Tn5-Linker and Linker-Tn5 separately into pACYCDuet-1. The following table shows an overview of what fragment(s) was/were cut with what restriction enzyme(s).

Component	Volume (uL)	Component	Volume (uL)	Component	Volume (uL)
10x CutSmart buffer	2	10x CutSmart buffer	2	10x CutSmart buffer	2
Fragment (Tn5-Linker)	10 (2000 ng)	Fragment (Tn5)	8 (2000 ng)	Fragment (Linker-Tn5)	9 (2000 ng)
Enzyme (FseI)	1	Enzyme (EcoRI)	1	Enzyme (EcoRI)	1
Enzyme (KpnI)	1	Enzyme (KpnI)	1	Enzyme (NotI)	1
MilliQ	6	MilliQ	8	MilliQ	7

Component	Volume (uL)	Component	Volume (uL)	Component	Volume (uL)
10x CutSmart buffer	2	10x CutSmart buffer	2	10x CutSmart buffer	2
Fragment (pACYCDuet-1)	16 (2000 ng)	Fragment (pACYCDuet-1)	16 (2000 ng)	Fragment (pACYCDuet-1)	16 (2000 ng)
Enzyme (FseI)	1	Enzyme (EcoRI)	1	Enzyme (EcoRI)	1
Enzyme (KpnI)	1	Enzyme (KpnI)	1	Enzyme (NotI)	1
MilliQ	0	MilliQ	0	MilliQ	0

The samples were incubated for 1 hour at 37 °C, and followed by 20 minute heat inactivation (65 °C) of the restriction enzyme(s).

Date

20/06/2018

Operator

Nicole Bennis

PCR Clean-Up

20 μL of each restriction reaction mixture was subjected to PCR Clean-Up, according to the protocol. Elution was done in 20 µL Milli-Q.

Date

20/06/2018

Operator

Nicole Bennis

Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

Isolate	Concentration (ng/uL)	260/280 ratio	260/230 ratio
pACYCDuet-1 cut with FseI and KpnI	18.7	1.71	1.02
pACYCDuet-1 cut with EcoRI and KpnI	41.2	1.80	1.44
pACYCDuet-1 cut with EcoRI and NotI	22.6	1.77	0.56
Tn5-Linker cut with FseI and KpnI	56.7	1.85	1.63
Tn5 cut with EcoRI and KpnI	59.2	1.84	1.20
Linker-Tn5 cut with EcoRI and NotI	70.0	1.87	1.73

Date

20/06/2018

Operator

Nicole Bennis

Ligation

In order to assemble cut fragments pACYCDuet-1 and Tn-Linker, Tn5 and Linker-Tn5 respectively, ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

DNA	Size (bp)	Cut with enzymes
Vector pACYCDuet-1	3970	EcoRI and NotI
Fragment Tn5-Linker	1490	EcoRI and NotI

DNA	Size (bp)	Cut with enzymes
Vector pACYCDuet-1	3768	KpnI and EcoRI
Fragment Tn5	1442	KpnI and EcoRI

DNA	Size (bp)	Cut with enzymes
Vector pACYCDuet-1	3984	FseI and KpnI
Fragment Linker-Tn5	1493	FseI and KpnI

A 20 μL ligation reaction mixture was prepared with the following composition:

Component	Volume (μL)	Component	Volume (μL)	Component	Volume (μL)
Ligase Buffer (10X)	2	Ligase Buffer (10X)	2	Ligase Buffer (10X)	2
T4 DNA Ligase	1	T4 DNA Ligase	1	T4 DNA Ligase	1
DNA vector (~200 ng)	10	DNA vector (~200 ng)	6	DNA vector (~200 ng)	11
DNA Tn5-Lin (~80 ng)	1.5	DNA Tn5 (~80 ng)	2	DNA Lin-Tn5 (~80 ng)	2
MilliQ	6.5	MilliQ	9	MilliQ	4

The samples were incubated for 4 hours at 20°C for ligation.

Date

20/06/2018

Operator

Nicole Bennis

Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

Strain	Plasmid and size (bp)	Cloning method	Volume added (μl)	Medium on which was plated
E.coli DH5α	pACYCDuet-1_Tn5-Lin (5460 bp)	Restriction/Ligation	4	LB-Cam
E.coli DH5α	pACYCDuet-1_Tn5 (5210 bp)	Restriction/Ligation	4	LB Cam
E.coli DH5α	pACYCDuet-1_Linker-Tn5 (5477 bp)	Restriction/Ligation	4	LB Cam
E.coli DH5α	pACYCDuet-1 restricted (neg. control)	Restriction	1	LB Cam
E.coli DH5α	pACYCDuet-1 (pos. control)	-	1	LB Cam

200 μl LB medium was added as recovery medium.

Date

21-06-2018

Operator

Nicole Bennis

Colony PCR

Colony PCR was done to verify propagation of pACYC1Duet-1_Lin-Tn5 after transformation from 20-06-2018. This plasmid contains the Lin-Tn5 insert, which can be checked with primers 029 and 030.

Template DNA	pACYCDuet-1_Lin-Tn5
Fw primer	029
Rv primer	030
expected amplicon size (bp)	1722 bp

23 tubes were prepared for colony PCR, containing pACYCDuet-1_Lin-Tn5 as template DNA. The thermocycler programme was installed to have an extension time of 2 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

Date

21-06-2018

Operator

Nicole Bennis

Liquid Starter Culture Preparation

Strain E.coli DH5a harbouring the pACYCDuet-1 plasmid was used for inoculation of 50 mL liquid starter culture (Luria Broth, complemented with chloramphenicol) following the Liquid Starter Culture protocol

Date

22-06-2018

Operator

Nicole Bennis

Colony PCR

Colony PCR was done to verify propagation of pACYC1Duet-1_Lin-Tn5 after transformation from 20-06-2018. This plasmid contains the Lin-Tn5 insert, which can be checked with primers 029 and 030.

Template DNA	pACYCDuet-1_Lin-Tn5
Fw primer	029
Rv primer	030
expected amplicon size (bp)	1722 bp

23 tubes were prepared for colony PCR, containing pACYCDuet-1_Lin-Tn5 as template DNA. The thermocycler programme was installed to have an extension time of 2 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

Date

21-06-2018

Operator

Nicole Bennis

DNA Gel Electrophoresis

The colony PCR performed on 21-06-2018 checking for pACYCDuet-1_Linker-Tn5 was run on a 0.8% agarose gel in TAE buffer. 4 μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

Unfortunately, no band was observed that corresponds to the Linker-Tn5 fragment in pACYCDuet-1. The bands are at the same height as the positive control pACYCDuet-1, meaning that no insertion has taken place

Date

27/06/2018

Operator

Nicole Bennis

Restriction

Restriction cloning was done for assembling Tn5, Tn5-Linker and Linker-Tn5 separately into pACYCDuet-1. The following table shows an overview of what fragment(s) were cut with what restriction enzymes.

Component	Volume (uL)	Component	Volume (uL)	Component	Volume (uL)
10x CutSmart buffer	2	10x CutSmart buffer	2	10x CutSmart buffer	2
Fragment (Tn5-Linker)	10 (2000 ng)	Fragment (Tn5)	8 (2000 ng)	Fragment (Linker-Tn5)	9 (2000 ng)
Enzyme (FseI)	1	Enzyme (EcoRI)	1	Enzyme (EcoRI)	1
Enzyme (KpnI)	1	Enzyme (KpnI)	1	Enzyme (NotI)	1
MilliQ	6	MilliQ	8	MilliQ	7

Component	Volume (uL)	Component	Volume (uL)	Component	Volume (uL)
10x CutSmart buffer	2	10x CutSmart buffer	2	10x CutSmart buffer	2
Fragment (pACYCDuet-1)	16 (2000 ng)	Fragment (pACYCDuet-1)	16 (2000 ng)	Fragment (pACYCDuet-1)	16 (2000 ng)
Enzyme (FseI)	1	Enzyme (EcoRI)	1	Enzyme (EcoRI)	1
Enzyme (KpnI)	1	Enzyme (KpnI)	1	Enzyme (NotI)	1
MilliQ	0	MilliQ	0	MilliQ	0

The samples were incubated for 1 hour at 37 °C, and followed by 20 minute heat inactivation (65 °C) of the restriction enzyme(s).

Date

20-06-2018

Operator

Nicole Bennis

PCR Clean-Up

20 μL of each restriction reaction mixture was subjected to PCR Clean-Up, according to the protocol. Elution was done in 30 µL Milli-Q.

Date

27/06/2018

Operator

Nicole Bennis

Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

Isolate	Concentration (ng/uL)	260/280 ratio	260/230 ratio
pACYCDuet-1 cut with FseI and KpnI	24.9	1.89	0.66
pACYCDuet-1 cut with EcoRI and NotI	20.8	1.81	1.08
Tn5-Linker cut with NotI and EcoRI	56.0	1.85	1.61
Linker-Tn5 cut with FseI and KpnI	53.4	1.88	1.57

Date

27/06/2018

Operator

Nicole Bennis

Ligation

In order to assemble cut fragments pACYCDuet-1 and Tn-Linker and Linker-Tn5 respectively, ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

DNA	Size (bp)	Cut with enzymes
Vector pACYCDuet-1	3970	EcoRI and NotI
Fragment Tn5-Linker	1490	EcoRI and NotI

DNA	Size (bp)	Cut with enzymes
Vector pACYCDuet-1	3984	FseI and KpnI
Fragment Linker-Tn5	1493	FseI and KpnI

A 20 μL ligation reaction mixture was prepared with the following composition:

Component	Volume (μL)	Component	Volume (μL)
Ligase Buffer (10X)	2	Ligase Buffer (10X)	2
T4 DNA Ligase	1	T4 DNA Ligase	1
DNA vector (~200 ng)	14.5	DNA vector (~200 ng)	14.5
DNA Tn5-Lin (~80 ng)	2.5	DNA Lin-Tn5 (~80 ng)	2.5
MilliQ	0	MilliQ	0

The samples were incubated overnight at 20°C for ligation.

Date

28/06/2018

Operator

Nicole Bennis

Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

Strain	Plasmid and size (bp)	Cloning method	Volume added (μl)	Medium on which was plated
E.coli DH5α	pACYCDuet-1_Tn5-Lin (5460 bp)	Restriction/Ligation	1.5 (~20 ng)	LB-Cam
E.coli DH5α	pACYCDuet-1_Tn5-Lin (5460 bp)	Restriction/Ligation	4 (~50 ng)	LB Cam
E.coli DH5α	pACYCDuet-1_Linker-Tn5 (5477 bp)	Restriction/Ligation	1.5 (~20 ng)	LB Cam
E.coli DH5α	pACYCDuet-1_Linker-Tn5 (5477 bp)	Restriction	4 (~50 ng)	LB Cam
E.coli DH5α	pACYCDuet-1 (pos. control)	-	0.1 (~10 ng)	LB Cam
E.coli DH5α	pACYCDuet-1 restricted (neg. control)	Restriction	2.5	LB Cam

200 μl LB medium was added as recovery medium.

Date

28/06/2018

Operator

Timothy Páez

Restriction

Restriction cloning was done for assembling Tn5-lin and lin-Tn5 into pACYCduet-1. The following table shows an overview of what fragments were cut with what restriction enzymes.
First Reaction:

Component	Volume (uL)
10x CutSmart buffer	2
Fragment (Tn5-lin)	9
Enzyme (ECORI HF)	1
Enzyme (NotI HF)	1
MilliQ	7

Component	Volume (uL)
10x CutSmart buffer	2
Vector (pACYC)	16
Enzyme (EcoRI HF)	1
Enzyme (NotI HF)	1
MilliQ	0

Second reaction:

Component	Volume (uL)
10x CutSmart buffer	2
Fragment (lin-Tn5)	10
Enzyme (FseI HF)	1
Enzyme (KpnI HF)	1
MilliQ	6

Component	Volume (uL)
10x CutSmart buffer	2
Vector (pACYC)	16
Enzyme (FseI HF)	1
Enzyme (KpnI HF)	1
MilliQ	0

The samples were incubated for 1 hour at 37 °C.

Date

28/06/2018

Operator

Timothy Páez

PCR clean-up

20 μL of restriction product were subjected to PCR Clean-Up, according to the protocol. Elution was done in 20 µL Milli-Q.

Products were quantified with Nanodrop, yielding concentration of:
- Tn5-lin: 32.3 ng/µL
- pACYC: 12.2 ng/µL
- lin-Tn5: 33.6 ng/µL
- pACYC: 11.2 ng/µL

Date

28/06/2018

Operator

Timothy Páez

Ligation

In order to assemble cut fragments Tn5-lin into pACYCduet-1 and lin-Tn5 into pACYCduet-1, ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

First reaction:

DNA	Size (bp)	Cut with enzymes
pACYCDuet	1.5 kbp	EcoRI and NotI
Tn5-lin	1.5 kbp	EcoRI and NotI

The ligation reaction mixture was prepared with the following composition

Component	Volume (uL)
Ligase Buffer (10x)	2 µL
T4 DNA Ligase	1 µL
pACYC (~100 ng)	7.35 µL
Tn5 (300 ng)	3.39 µL
MilliQ	6.26 µL

Second reaction:

DNA	Size (bp)	Cut with enzymes
pACYCDuet	1.5 kbp	FseI and KpnI
lin-Tn5	1.5 kbp	FseI and KpnI

The ligation reaction mixture was prepared with the following composition

Component	Volume (uL)
Ligase Buffer (10x)	2 µL
T4 DNA Ligase	1 µL
pACYC (~100 ng)	8.77 µL
lin-Tn5 (300 ng)	3.4 µL
MilliQ	4.8 µL

The samples were incubated overnight at 4 ºC for ligation.

Date

28/06/2018

Operator

Timothy Páez

Liquid Starter Culture Preparation

Escherichia coli DH5 alpha harbouring pACYC1-duet plasmid was used for inoculation of 10 mL liquid starter culture (Luria Broth, complemented with chloroamphenicol) following the Liquid Starter Culture Protocol.

Date

29/06/2018

Operator

Nicole Bennis

Colony PCR

Colony PCR was done to verify propagation of pACYC1Duet-1_Lin-Tn5 after transformation from 28-06-2018. This plasmid contains the Lin-Tn5 or Tn5-Lin insert, which can be checked with primers 029 and 030. There are two ways for colony PCR. The first one is picking a colony and directly resuspending the cells in PCR mixture. The second one is resuspending the colony in MilliQ, heating the suspension, centrifugation the broken cell pieces and taking the supernantant for PCR. In orde to test the efficiency of both methods, I tested both simultaneously. 22 colony PCR were performed by picking the colony and direct resuspension in the PCR mixture, while 20 colonies were picked and heated prior to pipetting the DNA into the PCR mixture.

Template DNA	pACYCDuet-1_Lin-Tn5
Fw primer	029
Rv primer	030
expected amplicon size (bp)	1722 bp

Template DNA	pACYCDuet-1_Tn5-Lin
Fw primer	029
Rv primer	030
expected amplicon size (bp)	1935 bp

The thermocycler programme was installed to have an extension time of 2 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

Date

29/06/2018

Operator

Timothy Páez

Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

Strain	Plasmid & size (bp)	Cloning method	Volume added (uL)	Medium on which was plated
E.coliDH5α	pACYC lin-Tn5 (3000 bp)	Restriction Ligation	10 µL	LB with Chloroamphenicol

400 µL LB medium was added as recovery medium.

Date

29/06/2018

Operator

Timothy Páez

Liquid Starter Culture Preparation

Escherichia coli DH5 alpha harbouring plasmid with dxCas9 from Addgene was used for inoculation of 10 mL liquid starter culture (Luria Broth, complemented with ampicillin) following the Liquid Starter Culture Protocol.

Date

30/06/2018

Operator

Timothy Páez

Colony PCR

Colony PCR was done to verify propagation of Escherichia coli DH5 alpha cells after transformation with pACYC containing lin-Tn5 (reaction 1) or Tn5-lin (reaction 2) as insert. This insertion can be checked with primers 029 and 030 (on T7 promoter and terminator).

Component
Template DNA	Colony solution
Forward primer	029
Reverse primer	030
Expected amplicon size (bp)	1500 bp

The number of colonies screened were:
- Tn5-lin: Colonies 1-6
- lin-Tn6: Colonies 8-10
- Control self ligation: Colony 11
The thermocycler programme was installed to have an extension time of 1 min 30 sec and an annealing temperature of 55 °C. After the programme was finished, samples were kept at 4°C.

Date

30/06/2018

Operator

Timothy Páez

DNA Gel Electrophoresis

Colony PCR products were run on a 0.8 % agarose gel in TAE buffer.
- 5 μL of PCR product was pipetted into each lane (no loading buffer required as PCR buffer contains loading buffer).
- 4 µL of 100 bp DNA ladder
- 80 V for 30 minutes
- The gel was imaged using a gel documentation system with UV-light.

Date

30/06/2018

Operator

Timothy Páez

Liquid Starter Culture Preparation

Escherichia coli DH5 alpha cells that possibly harbour positive pACYC1-duet plasmids with lin-Tn5 were used for inoculation of 10 mL liquid starter culture (Luria Broth, complemented with chloroamphenicol) following the Liquid Starter Culture Protocol.

Date

2/7/2018

Operator

Kavish Kohabir

Sequence Verification

Option 1: Sequence from plasmid

For verification of pTXB1-Tn5, primer FP030 was used for sequencing. The sequencing starts from the T7 terminator, in the reverse orientation respective to the Tn5 CDS. The 10µL sequencing sample was prepared in a 1.5 mL tube as follows:

Component	Volume (uL)	Final concentration
Plasmid DNA (500ng)	5.3	50 ng/uL
10µM primer 030	2.5	25 pmol
MilliQ	2.1	-

Within 7 days, the sequence was retrievable online and alignment with the in silico designed molecule showed 100% match of the 1050 bp downstream of the sequencing primer binding site.

Date

02/07/2018

Operator

Timothy Páez

Sequence Verification

Sequence from plasmid

For verification of Tn5-lin insertion in pACYC, primers 029 and 030 was used for sequencing. The sequencing starts from the -20 bp position of fragment Tn5-lin, flanking the promoter of T7 and terminator from T7 of the vector. Sequencing sample was prepared in 1.5 mL tubes for each primer as indicated my Macrogen for specific concentration of plasmid DNA.

Within 2 days, the sequence was retrievable online and alignment with the in silico designed molecule showed 100% match of the insert lin-Tn5, by aligning both primer sequences obtained.

Date

02/07/2018

Operator

Timothy Páez

High Fidelity PCR

To amplify dxCas9 (4 kbp), high fidelity PCR was done using Addgene plasmid harbouring dxCas9 as template DNA and 001 and 002 as forward and reverse primers respectively. Primer sequences can be found at: Primer sequences.

50 µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

The thermocycler programme was installed to have an extension time of 60 seconds and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

Date

02/07/2018

Operator

Timothy Páez

DNA Gel Electrophoresis

dxCas9 PCR product was run on a 0.8 % agarose gel in TAE buffer. 5μL was pipetted into each lane with 1 µL of loading dye. The gel was imaged using a gel documentation system with UV-light.

No amplification was observed. It is recomended to do PCR amplification again with longer extention time.

Date

3/7/2018

Operator

Kavish Kohabir

High Fidelity PCR

To amplify dxCas9 (4172bp), high fidelity PCR was done using dxCas(3.7)-VPR as template DNA and FP001 and FP002 as forward and reverse primers respectively. 6 25µL PCR mixtures were prepared, according to the High Fidelity PCR protocol. The thermocycler programme was installed to have an extension time of 2 minutes and an annealing temperature of 65 °C. After the programme was finished, samples were kept at 4°C.

Date

3/7/2018

Operator

Kavish Kohabir

DNA Gel Electrophoresis

dxCas9 PCR reaction products were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.
This gel shows that only samples 1, 2, 4 and 5 were successful attempts.

Date

02/07/2018

Operator

Timothy Páez

High Fidelity PCR

To amplify dxCas9 (4 kbp), high fidelity PCR was done using Addgene plasmid harbouring dxCas9 as template DNA and 001 and 002 as forward and reverse primers respectively. Primer sequences can be found at: Primer sequences.

50 µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

The thermocycler programme was installed to have an extension time of 2 minutes and an annealing temperature of 60 °C and 65 ºC. After the programme was finished, samples were kept at 4 °C.

Date

03/07/2018

Operator

Timothy Páez

DNA Gel Electrophoresis

PCR products of dxCas9 PCR were run on a 0.8 % agarose gel in TAE buffer.
- 5 μL of PCR product was pipetted into each lane with 1 µL of loading buffer.
- 4 µL of 500 bp DNA ladder
- 80 V for 30 minutes
- The gel was imaged using a gel documentation system with UV-light.

Date

04/07/2018

Operator

Timothy Páez

Restriction

Restriction cloning was done for assembling Cas9 into pACYCduet-1 and pACYC-duet lin-Tn5. The following table shows an overview of what fragments were cut with what restriction enzymes.
First Reaction (dxCas9 into pACYC):

Component	Volume (uL)
10x CutSmart buffer	2
Fragment (dxCas9)	15
Enzyme (NotI HF)	0.5
Enzyme (KpnI HF)	2.5
MilliQ	0

Component	Volume (uL)
10x CutSmart buffer	2
Vector (pACYC)	15
Enzyme (NotI HF)	0.5
Enzyme (KpnI HF)	2.5
MilliQ	0

Second reaction (dxCas9 into pACYC lin-Tn5):

Component	Volume (uL)
10x CutSmart buffer	2
Fragment (dxCas9)	15
Enzyme (NotI HF)	0.5
Enzyme (FseI HF)	2.5
MilliQ	0

Component	Volume (uL)
10x CutSmart buffer	2
Vector (pACYC lin-Tn5)	15
Enzyme (NotI HF)	0.5
Enzyme (FseI HF)	2.5
MilliQ	0

The samples were incubated for 1 hour at 37 °C.

Date

04/07/2018

Operator

Timothy Páez

PCR clean-up

20 μL of restriction product were subjected to PCR Clean-Up, according to the protocol. Elution was done in 20 µL Milli-Q.

Products were quantified with Nanodrop, yielding concentration of:
- pACYC: 12.7 ng/µL
- dxCas9: 40 ng/µL
- pACYC Tn5-lin: 9 ng/µL
- dxCas9: 31 ng/µL

Date

5/7/2018

Operator

Kavish Kohabir

High Fidelity PCR

To amplify Tn5-linker (1505bp), high fidelity PCR was done using pTXB1-Tn5 as template DNA and FP007 and FP008 as forward and reverse primers respectively. 3 100µL PCR mixtures were prepared, according to the High Fidelity PCR protocol. The thermocycler programme was installed to have an extension time of 1 minute and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

Date

5/7/2018

Operator

Kavish Kohabir

DNA Gel Electrophoresis

Tn5-Linker PCR reaction products were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light. The gel was not saved properly, but the results showed correct amplification of a ~1.5kb amplicon in sample 1 and 3.

Date

5/7/2018

Operator

Kavish Kohabir

PCR clean-up

200 μL of PCR product was subjected to PCR Clean-Up, according to the protocol. Elution was done in 60 µL Milli-Q.

Date

5/7/2018

Operator

Kavish Kohabir

Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

Isolate	Concentration (ng/uL)	260/280 ratio	260/230 ratio
Tn5-Linker	50	1.63	2.26

Date

5/7/2018

Operator

Kavish Kohabir

Restriction

Restriction cloning was done for assembling Tn5-Linker and dxCas9 into pACYC-Duet & for assembling dxCas9 into pACYC-Duet. The following table shows an overview of what fragments were cut with what restriction enzymes.

Component	Volume (µL)	Volume (µL)	Volume (µL)	Volume (µL)	Volume (µL)
10x CutSmart buffer	2	2	2	2	2
Fragment	10 (~500ng) (Tn5-Linker)	15 (~1.5µg)(dxCas9)	15 (~1.5µg)(dxCas9)	15.25 (~2µg) (pACYC-DUET)	15.25 (~2µg) (pACYC-DUET)
Enzyme	0.25 (EcoRI)	2.5 (FseI)	0.5 (NotI)	2.5 (FseI)	0.5 (NotI)
Enzyme	0.5 (NotI)	0.5 (NotI)	0.5 (KpnI)	0.25 (EcoRI)	0.5 (KpnI)
MilliQ	7.25	0	2	0	2

The samples were incubated for 1 hour(s) at 37 °C, and were not heat inactivated.

Date

5/7/2018

Operator

Kavish Kohabir

PCR clean-up

All restriction reaction mixtures were subjected to PCR Clean-Up, according to the protocol. Elution was done in 40 µL Milli-Q.

Date

5/7/2018

Operator

Kavish Kohabir

Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

Isolate	Concentration (ng/uL)	260/280 ratio	260/230 ratio
Tn5-Linker (cut with EcoRI & NotI)	6.5	1.74	2.26
dxCas9 (cut with NotI & FseI)	71	1.88	2.13
pACYC-Duet (cut with EcoRI & FseI)	24	1.76	2.19
dxCas9 (cut with NotI & KpnI)	84	1.77	2.22
pACYC-Duet (Cut with Noti & KpnI)	26	1.69	2.16

Date

5/7/2018

Operator

Kavish Kohabir

Ligation

In order to assemble cut Tn5-Linker and dxCas9 into pACYC-Duet, ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

DNA	Size (bp)	Cut with enzymes
Vector (pACYC-DUET)	3792	EcoRI & FseI
Fragment 1 (Tn5-Linker)	1490	EcoRI & NotI
Fragment 2 (dxCas9)	4172	FseI & NotI

A 20µL ligation reaction mixture was prepared with the following composition*

Component	Volume (µL)
Ligase Buffer (10x)	2
T4 DNA Ligase	1
DNA vector (~100 ng)	8
DNA fragment 1 (26 ng)	4
DNA fragment 2 (150 ng)	2.4
MilliQ	2.6

In order to assemble cut dxCas9 into pACYC-Duet, another ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

DNA	Size (bp)	Cut with enzymes
Vector (pACYC-DUET)	3806	KpnI & NotI
Fragment 1 (dxCas9)	4161	KpnI & NotI

A 20µL ligation reaction mixture was prepared with the following composition*

Component	Volume (µL)
Ligase Buffer (10x)	2
T4 DNA Ligase	1
DNA vector (~100 ng)	2.2
DNA fragment 1 (26 ng)	2
MilliQ	12.8

*For both ligations, a negative control was included separately (in which the insert fragment was substituted with equivalent volume of milli-Q).
All samples were incubated overnight on the bench (room temperature) for ligation.

Date

05/07/2018

Operator

Timothy Páez

Transformation of chemically competent cells

Using the ligate reactions from 04/07/2018, the following transformations were carried out according to the protocol for transformation of chemically competent cells:

Strain	Plasmid & size (bp)	Cloning method	Volume added (uL)	Medium on which was plated
E.coliDH5αlpha	pACYC dxCas9-lin-Tn5 (7500 bp) pACYC dxCas9 (5500 bp)	Restriction Ligation	10 µL	LB with Chloroamphenicol

400 µL LB medium was added as recovery medium.

Date

6/7/2018

Operator

Kavish Kohabir

Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

Strain	Plasmid & size (bp)	Cloning method	Volume added (uL)	Medium on which was plated
E.coliDH5α	pACYC-Tn5Linker-dxCas9 (9419bp)	Restriction-ligation	2.78 (~50ng)	LB+Cam
E.coliDH5α	pACYC-dxCas9 (7967bp)	Restriction-ligation	4.6 (~50ng)	LB+Cam
E.coliDH5α	3 negative controls (2x ligate without insert, 1x plain DH5α)	Restriction-ligation	20	LB+Cam
E.coliDH5α	pACYC-DUET (pos. ctrl)	-	3	LB+Cam

500 μl LB medium was added as recovery medium. 1x and 10x dilutions were plated for each transformation.

Date

06/07/2018

Operator

Timothy Páez

Colony PCR

Colony PCR was done to verify propagation of Escherichia coli DH5 alpha cells after transformation with pACYC containing dxCas9 (reaction 1) or pACYC containing dxCas9-lin-Tn5 (reaction 2) as insert. This insertion can be checked with primers 029 and 030 (on T7 promoter and terminator). Primer sequences can be found at: Primer sequences.

Component
Template DNA	Colony solution
Forward primer	029
Reverse primer	030
Expected amplicon size (bp)	(R1) 4000 bp (R2) 6000 bp

The number of colonies screened were:
- Cas9-lin-Tn5: Colonies 1-17
- Cas9: Colonies 18-45
- Control lin-Tn5: Colonies 46-68
- Control pACYC: Colonies 69-70
The thermocycler programme was installed to have an extension time of 6 min and an annealing temperature of 55 °C. After the programme was finished, samples were kept at 4°C.

Date

06/07/2018

Operator

Timothy Páez

DNA Gel Electrophoresis

Colony PCR products were run on a 0.8 % agarose gel in TAE buffer.
- 5 μL of PCR product was pipetted into each lane (no loading buffer required as PCR buffer contains loading buffer).
- 4 µL of 500 bp DNA ladder
- 80 V for 30 minutes
- The gel was imaged using a gel documentation system with UV-light.

Date

09/07/2018

Operator

Nicole Bennis

Restriction

In our laboratory, we experienced some trouble with restriction ligation cloning. Therefore, I aimed for checking the efficiency of all restriction enzymes used in the cloning process. I used the pACYCDuet-1 plasmid and restricted this plasmid with the restriction enzyme XbaI and one of the restriction enzymes EcoRI, KpnI, NotI or FseI. To compare, I restricted the pACYCDuet-1 plasmid only with XbaI and as negative control I used no restriction enzyme to cut pACYCDuet-1.

The following table shows an overview of what fragment(s) were cut with what restriction enzyme(s).

Component	Volume (uL)	Component	Volume (uL)	Component	Volume (uL)
10x CutSmart buffer	2	10x CutSmart buffer	2	10x CutSmart buffer	2
pACYCDuet-1	10 (~1000 ng)	pACYCDuet-1	10 (~1000 ng)	pACYCDuet-1	10 (~1000 ng)
Enzyme (XbaI)	0.5	Enzyme (XbaI)	0.5	Enzyme (none)	0
Enzyme (EcoRI, KpnI, FseI or NotI)	0.5	Enzyme (none)	0	Enzyme (none)	0
MilliQ	7	MilliQ	7.5	MilliQ	8

The samples were incubated for 4 hour(s) at 37 °C, and followed by 20 minute heat inactivation of the restriction enzyme(s).

Date

09/07/2018

Operator

Nicole Bennis

DNA Gel Electrophoresis

The restriction efficiency experiment performed on 09/07/2018 checking for activity of NotI, EcoRI, FseI and KnpI was run on a 0.8% agarose gel in TAE buffer. 4 μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

From the DNA gel electrophoresis, it can be concluded that the restriction enzymes work efficiently for restriction ligation cloning purposes.

Date

9/7/2018

Operator

Kavish Kohabir

Transformation Results

After carrying out the transformation protocol on 6/7/2018, the plates were left for overnight incubation. After ~14-18hr incubation, the plates were stored at 4°C until 9/7/2018 for observing the results

Strain	DNA	Medium	Result
E.coli DH5α	pACYC-Tn5Linker-dxCas9 (9419bp)	LB+Cam	20 colonies (1x diluted) 4 colonies (10x diluted)
E.coli DH5α	pACYC-dxCas9 (7967bp)	LB+Cam	3 colonies (1x diluted) No colonies (10x diluted)
E.coli DH5α	None (all neg. controls)	LB+Cam	no colonies
E.coli DH5α	pACYC-Duet (pos. ctrl.)	LB+Cam	>500 colonies

Date

9/7/2018

Operator

Kavish Kohabir

Colony PCR

Colony PCR was done to verify propagation of pACYC-Tn5Linker-dxCas9 (9419bp) and pACYC-dxCas9 (7967bp) after transformation on 6/7/2018. These plasmids contain the T7 expression system, which allow primers FP029 and FP030 to anneal.

Template DNA	pACYC-Tn5Linker-dxCas9	pACYC-dxCas9
FW primer	029	029
RV primer	030	030
Expected amplicon size (bp)	5894	4442

27 tubes were prepared for colony PCR, containing picked colony as template DNA. pACYC was used as positive control template. The thermocycler programme was installed to have an extension time of 6 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

Date

9/7/2018

Operator

Kavish Kohabir

DNA Gel Electrophoresis

All colony PCR samples were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light. None of the reactions, not even the positive control showed presence of amplicon.

Date

09/07/2018

Operator

Timothy Páez

Sequence Verification

Sequence from plasmid

For verification of Tn5-lin insertion in pACYC, primers 003, 012, 029 and 030 were used for sequencing. The sequencing starts from the -20 bp position of fragment Tn5-lin, flanking the promoter of T7 and terminator from T7 of the vector. Sequencing sample was prepared in 1.5 mL tubes for each primer as indicated my Macrogen for specific concentration of plasmid DNA.
Primer sequences can be found at: Primer sequences.

Within 2 days, the sequence was retrievable online and alignment with the in silico designed molecule showed 100% match of the insert lin-Tn5, by aligning both primer sequences obtained.

Date

10/07/2018

Operator

Nicole Bennis

Restriction

Restriction cloning was done for assembling Tn5-Linker and dxCas9 separately into pACYCDuet-1 or pACYCDuet-1_Lin-Tn5, respectively. The following table shows an overview of what fragment(s) were cut with what restriction enzymes.

Component	Volume (uL)	Component	Volume (uL)
10x CutSmart buffer	5	10x CutSmart buffer	5
pACYCDuet-1	41 (2000 ng)	Fragment (Tn5-Lin)	41 (2000 ng)
Enzyme (NotI)	2	Enzyme (NotI)	2
Enzyme (EcoRI)	2	Enzyme (EcoRI)	2
MilliQ	0	MilliQ	0

Component	Volume (uL)	Component	Volume (uL)
10x CutSmart buffer	5	10x CutSmart buffer	5
pACYCDuet-1_Lin-Tn5	41 (2000 ng)	Fragment (dxCas9)	41 (2000 ng)
Enzyme (NotI)	2	Enzyme (NotI)	2
Enzyme (FseI)	2	Enzyme (FseI)	2
MilliQ	0	MilliQ	0

The samples were incubated overnight at 37 °C and followed by 20 minute heat inactivation (65 °C) of the restriction enzymes.

Date

10/07/2018

Operator

Nicole Bennis

PCR Clean-Up

20 μL of each restriction reaction mixture was subjected to PCR Clean-Up, according to the protocol. Elution was done in 30 µL Milli-Q.

Date

10/07/2018

Operator

Nicole Bennis

Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

Isolate	Concentration (ng/uL)	260/280 ratio	260/230 ratio
Tn5-Lin cut with EcoRI and NotI	36.3	1.80	0.09
pACYCDuet-1 cut with EcoRI and NotI	53.6	1.88	0.17
dxCas9 cut with NotI and FseI	59.5	1.87	0.49
pACYCDuet-1_Lin-Tn5 cut with FseI and NotI	50.0	2.00	0.47

Date

10/07/2018

Operator

Nicole Bennis

Ligation

In order to assemble cut fragments pACYCDuet-1 with Tn-Linker and pACYCDuet-1_Lin-Tn5 with dxCas9 ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

DNA	Size (bp)	Cut with enzymes
Vector pACYCDuet-1	3970	EcoRI and NotI
Fragment Tn5-Linker	1490	EcoRI and NotI

DNA	Size (bp)	Cut with enzymes
Vector pACYCDuet-1_Lin-Tn5	5299	FseI and NotI
Fragment dxCas9	4150	FseI and NotI

A 20 μL ligation reaction mixture was prepared with the following composition:

Component	Volume (μL)	Component	Volume (μL)
Ligase Buffer (10X)	2	Ligase Buffer (10X)	2
T4 DNA Ligase	1	T4 DNA Ligase	1
DNA vector (~200 ng)	5	DNA vector (~200 ng)	5
DNA Tn5-Lin (~100 ng)	5	DNA dxCas9 (~600 ng)	12
MilliQ	7	MilliQ	0

The samples were incubated for 8 hours at 4°C for ligation.

Date

20-06-2018

Operator

Nicole Bennis

Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

Strain	Plasmid and size (bp)	Cloning method	Volume added (μl)	Medium on which was plated
E.coli DH5α	pACYCDuet-1_Tn5-Lin (5460 bp)	Restriction/Ligation	4	LB-Cam
E.coli DH5α	pACYCDuet-1_dxCas9-Linker-Tn5 (9449 bp)	Restriction/Ligation	4	LB Cam
E.coli DH5α	pACYCDuet-1 restricted (neg. control)	Restriction	1	LB Cam
E.coli DH5α	pACYCDuet-1 (pos. control)	-	1	LB Cam

200 μl LB medium was added as recovery medium.

Date

10/7/2018

Operator

Kavish Kohabir

Colony PCR

Colony PCR was done to verify propagation of pACYC-Tn5Linker-dxCas9 (9419bp) and pACYC-dxCas9 (7967bp) after transformation on 6/7/2018. These plasmids contain the T7 expression system, which allow primers FP029 and FP030 to anneal.

Template DNA	pACYC-Tn5Linker-dxCas9	pACYC-dxCas9
FW primer	029	029
RV primer	030	030
Expected amplicon size (bp)	5894	4442

27 tubes were prepared for colony PCR, containing boiled colony as template DNA (colonies were picked from the restreaked plate on 9/7/2018). Vector pACYC was used as positive control template. The thermocycler programme was installed to have an extension time of 6 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

Date

10/7/2018

Operator

Kavish Kohabir

DNA Gel Electrophoresis

All colony PCR samples were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.
Colonies 11, 13, 20, 21, 22, 23, 24 and 25 show vague smears around the expected height (Tn5LCas = 5894bp)

Date

10/7/2018

Operator

Kavish Kohabir

Liquid Starter Culture Preparation

Colonies 11, 13, 20, 21, 22, 23, 24 and 25 (all potentially pACYC-Tn5Linker-dxCas9) were used for inoculation of 5mL liquid starter culture (LB+Cam) each, following the Liquid Starter Culture Protocol.

Date

11/07/2018

Operator

Nicole Bennis

Restriction

In our laboratory, we experienced some trouble with restriction ligation cloning. Therefore, will use a different restriction buffer: the universal buffer of JenaBioscience. Also, I will try to ligate the Tn-Linker and Linker-Tn5 separately to the dxCas9 prior to cloning into pACYCDuet-1. The following table shows an overview of what fragment(s) were cut with what restriction enzyme(s).

Component	Volume (uL)	Component	Volume (uL)
10x Universal buffer	2	10x Universal buffer	2
dxCas9	10 (~2000 ng)	Tn5-Linker	10 (~2000 ng)
Enzyme (NotI)	0.5	Enzyme (NotI)	0.5
MilliQ	7.5	MilliQ	7.5

Component	Volume (uL)	Component	Volume (uL)
10x Universal buffer	2	10x Universal buffer	2
dxCas9	10 (~2000 ng)	Linker-Tn5	10 (~2000 ng)
Enzyme (FseI)	0.5	Enzyme (FseI)	0.5
MilliQ	7.5	MilliQ	7.5

The samples were incubated for 4 hour(s) at 37 °C, and followed by 20 minute heat inactivation of the restriction enzyme(s).

Date

11/07/2018

Operator

Nicole Bennis

Colony PCR

Colony PCR was done to verify propagation of pACYC1Duet-1_Tn5-Lin after transformation from 10/07/2018. This plasmid contains the Tn5-Lin insert, which can be checked with primers 029 and 030. Also, the pACYC1Duet-1_dxCas9-Lin-Tn5 transformation resulted in colonies, which were also checked. However, on the negative control transformation plate with restricted pACYCDuet-1, colonies were observed.

Template DNA	pACYCDuet-1_Tn5-Lin
Fw primer	029
Rv primer	030
expected amplicon size (bp)	1935 bp

Template DNA	pACYCDuet-1_dxCas9-Lin-Tn5
Fw primer	029
Rv primer	030
expected amplicon size (bp)	5924 bp

15 tubes were prepared for colony PCR, containing pACYCDuet-1_dxCas9-LinTn5 as template DNA. Additionally, 14 tubes were prepared for colony PR containing pACYCDuet-1_Tn5-Lin as template DNA. For positive control, 4 tubes for colonies with pACYCDuet-1 were used. The thermocycler programme was installed to have an extension time of 6 and 4 minutes respectively and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

Date

11/7/2018

Operator

Kavish Kohabir

Plasmid Isolation

Overnight starter cultures of colonies 11, 13, 19, 20, 21, 22, 23, and 24 (all potentially pACYC-Tn5Linker-dxCas9) were subjected to plasmid isolation following the Plasmid Isolation Protocol. Final elution was done with 30 μL of pre-warmed MilliQ.

Date

11/7/2018

Operator

Kavish Kohabir

Nanodrop DNA Quantification

Plasmid isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentrations were measured and verified to be higher than 10ng/µl. The purity of the DNA (260/280 and 260/230 ratios) was also checked to be approximately 1.8~1.9.

Date

11/7/2018

Operator

Kavish Kohabir

Colony PCR

Diagnostic PCR on isolated plasmids from inoculated colonies was done as attempt to verify propagation of pACYC-Tn5Linker-dxCas9 (9419bp) after transformation on 6/7/2018. This plasmids contains the T7 expression system, which allow primers FP029 and FP030 to anneal.

Template DNA	pACYC-Tn5LinkerdxCas9
Fw primer	029
Rv primer	030
Expected amplicon size (bp)	5894

8 tubes were prepared for colony PCR, containing isolated plasmid as template DNA (isolated from colonies inoculated on 10/7/2018). Vector pACYC was used as positive control template. The thermocycler programme was installed to have an extension time of 6.5 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

Date

11/7/2018

Operator

Kavish Kohabir

DNA Gel Electrophoresis

Colony PCR samples were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.
This gel showed all of the plasmid isolate templates result in double bands, which is corresponding to the pACYC-DUET control (lane 9). Compared to the Tn5 insert (lane 10), this indicates all of the plasmid isolates are pACYC-DUET and contain no insert.

Date

12/07/2018

Operator

Nicole Bennis

Colony PCR

Colony PCR was done to verify propagation of pACYC1Duet-1_Lin-Tn5 after transformation from 11/07/2018. This plasmid contains the Lin-Tn5 insert, which can be checked with primers 029 and 030.

Template DNA	pACYCDuet-1_Lin-Tn5
Fw primer	029
Rv primer	030
expected amplicon size (bp)	1722 bp

23 tubes were prepared for colony PCR, containing pACYCDuet-1_Lin-Tn5 as template DNA. The thermocycler programme was installed to have an extension time of 2 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

Date

12/06/2018

Operator

Nicole Bennis

DNA Gel Electrophoresis

The colony PCR performed on 11-07-2018 checking for pACYCDuet-1_Tn5-Linker and pACYCDuet-1_dxCas9-Lin-Tn5 was run on a 0.8% agarose gel in TAE buffer. 4 μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

Unfortunately, no band was observed that corresponds to the dxCas9 fragment in pACYCDuet-1_Linker-Tn5. Also, no band was observed that indicates integration of Tn5-Lin into pACYCDuet. The bands are at the same height as the positive control pACYCDuet-1, meaning that no insertion has taken place.

Date

12/7/2018

Operator

Kavish Kohabir

Restriction

Restriction cloning was done for assembling Tn5-Linker and dxCas9 into pACYC-Duet & for assembling dxCas9 and Linker-Tn5 into pACYC-Duet. The following table shows an overview of what fragments were cut with what restriction enzymes.

Component	Volume (µL)	Volume (µL)	Volume (µL)	Volume (µL)	Volume (µL)
10x CutSmart buffer	5	5	5	5	5
Fragment	5 (~1µg) (Tn5-Linker)	14 (~1µg)(dxCas9)	25 (~1µg)(pACYC-DUET)	5 (~2µg) (Linker-Tn5)	25 (~1µg) (pACYC-DUET)
Enzyme	0.5 (EcoRI)	1 (FseI)	1 (FseI)	1 (FseI)	0.5 (NotI)
Enzyme	0.5 (NotI)	0.5 (NotI)	0.25 (EcoRI)	0.5 (KpnI)	0.5 (KpnI)
MilliQ	39	29.5	18.5	38.5	19

The samples were incubated for 4 hour(s) at 37 °C, and were not heat inactivated.

Date

12/7/2018

Operator

Kavish Kohabir

PCR clean-up

Restriction products from 12/7/2018 were subjected to PCR Clean-Up, according to the protocol. Elution was done in 30 µL Milli-Q.

Date

12/7/2018

Operator

Kavish Kohabir

Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

Isolate	Cut with	Concentration (ng/uL)	260/280 ratio	260/230 ratio
Tn5-Linker	EcoRI&NotI	48	1.9	1.1
Linker-Tn5	FseI&KpnI	30	2	0.9
dxCas9	FseI & NotI	8.2	2.2	0.3
pACYC-DUET	FseI&EcoRI	18	1.8	0.6
pACYC-DUET	FotI&KpnI	15	1.8	0.6

Date

12/7/2018

Operator

Kavish Kohabir

Ligation

In order to assemble cut Tn5-Linker and cut dxCas9 into cut pACYC-Duet, ligation was performed. This table gives an overview of which fragments were cut with which enzymes. the approach taken is a single-pot ligation, in which both inserts (dxCas9 and Tn5)

DNA	Size (bp)	Cut with enzymes
Vector (pACYC-DUET)	3792	EcoRI & FseI
Fragment 1 (Tn5-Linker)	1490	EcoRI & NotI
Fragment 2 (dxCas9)	4172	NotI & FseI

A 20 µl ligation reaction mixture was prepared with the following composition*

Component	Volume (uL)
Ligase Buffer (10x)	3
T4 DNA Ligase	1.5
DNA vector (~50 ng)	2.5
DNA fragment 1 (~50 ng)	1
DNA fragment 2 (~60 ng)	20
MilliQ	2

In order to assemble cut dxCas9 and cut Linker-Tn5 into cut pACYC-Duet, another ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

DNA	Size (bp)	Cut with enzymes
Vector (pACYC-DUET)	3806	NotI & KpnI
Fragment 1 (dxCas9)	4161	FseI & NotI
Fragment 2 (Linker-Tn5)	1508	FseI & KpnI

A 20 µl ligation reaction mixture was prepared with the following composition*

Component	Volume (uL)
Ligase Buffer (10x)	3
T4 DNA Ligase	1.5
DNA vector (~50 ng)	2.5
DNA fragment 1 (~50 ng)	20
DNA fragment 2 (~50 ng)	1.8
MilliQ	1.2

*For both ligations, a negative control was included separately (in which ONE of the insert fragments was substituted with equivalent volume of milli-Q).
All samples were incubated overnight on the bench (room temperature) for ligation.

Date

13/07/2018

Operator

Nicole Bennis

PCR Clean-Up

20 μL of each restriction reaction mixture done on 11/07/2018 was subjected to PCR Clean-Up, according to the protocol. Elution was done in 30 µL Milli-Q.

Date

13/07/2018

Operator

Nicole Bennis

Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

Isolate	Concentration (ng/uL)	260/280 ratio	260/230 ratio
Tn5-Lin cut with NotI	58.1	1.92	0.70
dxCas9 cut with NotI	72.3	1.84	1.68
Linker-Tn5 cut with FseI	59.5	1.88	1.34
dxCas9 cut with FseI	48.7	1.84	0.76

Date

13/07/2018

Operator

Nicole Bennis

Ligation

In order to assemble cut fragments dxCas9 with Tn-Linker and dxCas9 with Linker-Tn5 respectively, ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

DNA	Size (bp)	Cut with enzymes
dxCas9	4172	NotI
Fragment Tn5-Linker	1495	NotI

DNA	Size (bp)	Cut with enzymes
dxCas9	4157	FseI
Linker-Tn5	1508	FseI

A 20 μL ligation reaction mixture was prepared with the following composition:

Component	Volume (μL)	Component	Volume (μL)
Ligase Buffer (10X)	2	Ligase Buffer (10X)	2
T4 DNA Ligase	1	T4 DNA Ligase	1
DNA dxCas9 (~200 ng)	4	DNA dxCas9 (~200 ng)	4
DNA Tn5-Lin (~100 ng)	2	DNA Lin-Tn5 (~100 ng)	2
MilliQ	11	MilliQ	11

The samples were incubated for 2.5 hours at 4°C for ligation.

Date

19-06-2018

Operator

Nicole Bennis

High Fidelity PCR

To amplify the dxCas9-Linker-Tn5 (5655 bp) Tn5-Linker-dxCas9 (5660 bp) coding sequence from the ligation product, high fidelity PCR was done. The template DNA is the ligation mixture and the primers that are used are the combinations 001 & 006 and 002 & 007 respectively. Two 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.
Primer combination(s) and expected fragment(s)

Sample	Fw primer	Rv primer	Expected fragment	Expected size (bp)
1	001	006	dxCas9-Linker-Tn5	5655
2	002	007	Tn5-Linker-dxCas9	5660

The thermocycler programme was installed to have an extension time of 6 min 30 seconds and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

Date

13/7/2018

Operator

Kavish Kohabir

DNA Gel Electrophoresis

The restriction products from 11/7/2018 were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.
This gel shows that one of the dxCas9 samples (lane 3) was not purified.

Date

13/7/2018

Operator

Kavish Kohabir

Diagnostic PCR

Diagnostic PCR on ligation samples from 12/7/2018 were done to check for presence of correct constructs. Primer sets were chosen based on unique binding opportunities either to pACYC-Duet, dxCas9 and/or Tn5. The expected bands were estimated based on in silico design.

Template DNA	pACYC-Tn5Linker-dxCas9				pACYC-dxCas9-LinkerTn5
Fw primer	029	007	001	029	029	001	007	029
Rv primer	030	002	030	006	030	006	030	002
Expected amplicon size (bp)	5907	5660	4295	1576	5924	5655	1561	4332

The thermocycler programme was installed to have an extension time of 6.5 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C

Date

13/7/2018

Operator

Kavish Kohabir

Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

Strain	Plasmid & size (bp)	Cloning method	Volume added (µL)	Medium
E.coliDH5α	pACYC-Tn5Linker-dxCas9 (~10kb)	Restriction-ligation	20µL	LB+Cam
E.coliDH5α	pACYC-dxCas9-LinkerTn5 (~10kb)	Restriction-ligation	20µL	LB+Cam
E.coliDH5α	pACYC-dxCas9-LinkerTn5 (~10kb)	Restriction-ligation	-	LB+Cam

500 μl LB medium was added as recovery medium. 1x and 10x dilutions were plated for each transformation.

Date

16/07/2018

Operator

Nicole Bennis

DNA Gel Electrophoresis

The ligation performed on 13/07/2018 for construction of Tn5-Linker-dxCas9 and dxCas9-Lin-Tn5 was run on a 0.8% agarose gel in TAE buffer. 4 μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

The ligation reaction resulted in the expected sizes of the dxCas9-Linker-Tn5 (5655 bp) Tn5-Linker-dxCas9 (5660 bp) coding sequence.

Date

16/07/2018

Operator

Nicole Bennis

High Fidelity PCR

To amplify dxCas9-Linker-Tn5 (5655 bp) Tn5-Linker-dxCas9 (5660 bp), high fidelity PCR was done using the ligation reactions as template DNA ausing the forward and reverse primers sets 001 & 007 and 002 & 006 respectively. 2 times 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.
Primer combination(s) and expected fragment(s)

Sample	Fw primer	Rv primer	expected fragment	expected size (bp)
1	001	006	dxCas9-Linker-Tn5	5655
2	002	007	Tn5-Linker-dxCas9	5660

The thermocycler programme was installed to have an extension time of 60 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

Date

16/07/2018

Operator

Nicole Bennis

DNA Gel Electrophoresis

To amplify the ligation products Tn5-Linker-dxCas9 and dxCas9-Lin-Tn5 a High Fidelity PCR was performed and the result was run on a 0.8% agarose gel in TAE buffer. 4 μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

The PCR reaction on the ligation products resulted in the expected sizes of the dxCas9-Linker-Tn5 (5655 bp) Tn5-Linker-dxCas9 (5660 bp) coding sequence in large amounts of DNA. However, there is also one other band of DNA present, indicating an off-target amplicon generated during the PCR.

Date

16-07-2018

Operator

Nicole Bennis

DNA Gel Excision and Purification

Due to presence of off-target amplicons, the PCR mixture is again loaded on a gel. Subsequently, the required fragments containing Tn5-Linker-dxCas9 and dxCas9-Lin-Tn5 were excised. The agarose slice containing the desired DNA were purified by a DNA Clean-Up, according to the protocol. Elution was done in 30 µL prewarmed Milli-Q.

Date

16/07/2018

Operator

Nicole Bennis

Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

Isolate	Concentration (ng/uL)	260/280 ratio	260/230 ratio
Tn5-Linker-dxCas9	37.0	1.32	0.77
dxCas9-Lin-Tn5	35.9	1.87	0.52

Date

16/07/2018

Operator

Nicole Bennis

High Fidelity PCR

To amplify dxCas9, Linker-Tn5 (1508 bp) and Tn5-Linkr (1505 bp), high fidelity PCR was done. The forward and reverse primers sets that were used are 001 & 002 and 005 & 006 and 007 & 008 respectively. Two times 3 times 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol. However, one of the sets of 3 times 50µL PCR mixtures uses HF buffer and Phusion Polymerase, while the other set of 3 times 50µL PCR mixtures uses Q5 buffer and Q5 Polymerase.
Primer combination(s) and expected fragment(s)

Sample	Fw primer	Rv primer	Expected fragment	Expected size (bp)
1	001	002	dxCas9	4172
2	005	006	Linker-Tn5	1508
3	007	008	Tn5-Linker	1505

The thermocycler programme was installed to have an extension time of 6 minutes and 30 seconds and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

Date

16/07/2018

Operator

Nicole Bennis

DNA Gel Electrophoresis

The amplicons generated during the High Fidelity PCR were run on a 0.8% agarose gel in TAE buffer. 4 μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

The PCR reaction o resulted in the expected sizes of the dxCas9 (4172 bp) Tn5-Linker (1505 bp) and Linker-Tn5 (1508 bp) coding sequence in large amounts of DNA.

Date

16/07/2018

Operator

Nicole Bennis

Liquid Starter Culture Preparation

E.coli strains harbouring the plasmid pACYCDuet-1 and pACYCDuet-1_Lin-Tn5 were used for inoculation of 10 mL liquid starter culture (Luria Broth, complemented with ampicillin) following the Liquid Starter Culture Protocol.

Date

16/7/2018

Operator

Kavish Kohabir

DNA Gel Electrophoresis

Diagnostic PCR reaction products from 13/7/2018 were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.
This gel shows that partially, the constructs have been ligated (e.g. lane 8 & 9), but the complete plasmid does not seem to be constructed (lanes 2 & 6).

Date

17/07/2018

Operator

Nicole Bennis

Plasmid Isolation

Overnight starter cultures of pACYCDuet-1_Lin-Tn5 and pACYCDuet-1 were subjected to plasmid isolation following the Plasmid Isolation Protocol. Final elution was done with 30 μL of pre-warmed MilliQ.

Date

17/07/2018

Operator

Nicole Bennis

PCR Clean-Up

50 μL of each PCR reaction done on 16/07/2018 was subjected to PCR Clean-Up, according to the protocol. Elution was done in 30 µL Milli-Q.

Date

17-07-2018

Operator

Nicole Bennis

Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

Isolate	Concentration (ng/uL)	260/280 ratio	260/230 ratio
pACYCDuet-1	7.1	2.19	1.23
pACYCDuet-1	7.9	1.81	1.19
pACYCDuet-1	10.7	1.52	0.77
pACYCDuet-1_Lin-Tn5	15.5	1.69	0.89
pACYCDuet-1_Lin-Tn5	10.1	1.59	0.99
pACYCDuet-1_Lin-Tn5	10.2	1.64	0.97
Q5: Lin-Tn5	180.9	1.80	1.52
Q5: Tn5-Lin	148.3	1.80	1.92
Q5: dxCas9	259.6	1.84	1.82
Phusion: Lin-Tn5	183.8	1.85	1.86
Phusion: Tn5-Lin	164.1	1.85	1.86
Phusion: dxCas9	233.9	1.83	1.77

Date

17/7/2018

Operator

Kavish Kohabir

High Fidelity PCR

The following high fidelity PCR reactions were carried out:

Amplicon	Size (bp)	Fw primer	Rv primer	template	Elongation time (sec)	Annealing temperature (°C)
LacZ-gRNA	314	IV003	IV004	Gblock	60	60
KanR CDS	862	IV005	IV006	pSB1K3	60	60
dxCas9	~4200	001	002	dxCas9(3.7)-VPR	240	60
Tn5-Linker	~1500	007	008	pACYC-Tn5Linker	240	60
Linker-Tn5	~1500	005	006	pACYC-LinkerTn5	240	60
dxCas9-LinkerTn5	~6000	001	006	dxCas9 & Tn5	240	60
Tn5Linker-dxCas9	~6000	007	002	dxCas9 & Tn5	240	60

PCR mixtures were prepared, according to the High Fidelity PCR protocol. After the programme was finished, samples were kept at 4°C.

Date

17/7/2018

Operator

Kavish Kohabir

PCR clean-up

PCR products were subjected to PCR Clean-Up, according to the protocol. Elution was done in 50 µL Milli-Q.

Date

17/7/2018

Operator

Kavish Kohabir

Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

Isolate	260/280 ratio	260/230 ratio	Concentration (ng/uL)
LacZ-gRNA			85
KanR cassette			209
dxCas9			128
Tn5Linker			162
LinkerTn5	1.87	2.02	181
dxCas9Linker-Tn5	1.82	1.88	74.8
Tn5Linker-dxCas9	1.81	1.91	78.7
dxCas9Tn5+upstr EcoRI	1.79	1.81	56.8
Tn5dxCas9 + upstr EcoRI	1.8	1.99	89.4
dxCas9Tn5 + downstr EcoRI	1.85	1.93	109
Tn5dxCas9 + downstr EcoRI	1.82	1.99	101.4
dxCas9	1.85	2.11	138

Date

17/7/2018

Operator

Kavish Kohabir

Restriction

Restriction cloning was done for assembling Tn5-Linker and dxCas9 into pACYC-Duet & for assembling dxCas9 into pACYC-Duet. The following table shows an overview of what fragments were cut with what restriction enzymes.

Component	Volume (uL)	Volume (uL)	Volume (uL)	Volume (uL)	Volume (uL)
10x CutSmart buffer	5	5	5	5	5
Fragment	43 (pACYC-LinkerTn5)	10 (dxCas9)	43 (pACYC-LinkerTn5)	15 (Tn5-Linker)	10 (dxCas9)
Enzyme	1 (NotI)	1 (FseI)	1 (EcoRI)	1 (EcoRI)	1 (NotI)
Enzyme	1 (FseI)	1 (NotI)	1 (KpnI)	1 (NotI)	1 (KpnI)
MilliQ	0	0	0	28	33

Component	Volume (uL)	Volume (uL)	Volume (uL)	Volume (uL)	Volume (uL)
10x CutSmart buffer	5	5	5	5	5
Fragment	43 (pACYC-DUET)	25 (Tn5Linker-dxCas9)	15 (Tn5-Linker)	43 (pACYC-DUET)	10 (dxCas9-LinkerTn5)
Enzyme	1 (NotI)	1 (FseI)	1 (EcoRI)	1 (EcoRI)	1 (NotI)
Enzyme	1 (FseI)	1 (NotI)	1 (KpnI)	1 (NotI)	1 (KpnI)
MilliQ	0	0	0	28	33

The samples were incubated for 4 hour(s) at 37 °C, and were not heat inactivated.

Date

17/7/2018

Operator

Kavish Kohabir

PCR clean-up

Restriction products were subjected to PCR Clean-Up, according to the protocol. Elution was done in 50 µL Milli-Q.

Date

17/7/2018

Operator

Kavish Kohabir

Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

Fragment	Cut with	260/280 ratio	260/230 ratio	Concentration (ng/uL)
pACYC-LinkerTn5	NotI & FseI	1.75	0.69	8
dxCas9	NotI & FseI	1.85	1.51	2.37
pACYC	EcoRI & KpnI	1.88	1.05	10.6
Tn5Linker	NotI & EcoRI	1.82	1.33	26.1
dxCas9	NotI & KpnI	1.86	1.51	29.6
pACYC-DUET	NotI & KpnI	1.81	1.03	13.8
dxCas9	NotI & FseI	1.84	1.19	20.8
Linker-Tn5	FseI & KpnI	1.84	1.54	42
pACYC-DUET	NotI & KnpI	1.79	1.06	13.1
dxCas9-LinkerTn5	NotI & KnpI	1.79	0.54	5.5
pACYC-DUET	EcoRI & KpnI	1.67	0.97	13
Tn5Linker-dxCas9	EcoRI & KpnI	2	0.78	6.1

Date

17/7/2018

Operator

Kavish Kohabir

Ligation

In order to assemble cut fragments, ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

A 50µl ligation reaction mixture was prepared with the following composition

Component	L1* Volume (uL)	L2* Volume (uL)	L3* Volume (uL)	L4* Volume (uL)	L5* Volume (uL)
Ligase Buffer (10x)	5	5	5	5	5
T4 DNA Ligase	2.5	2.5	2.5	2.5	2.5
DNA vector	24 (pACYC-LinkerTn5, ~200ng)	17 (pACYC, ~200ng)	12.5 (pACYC, ~150ng)	4 (pACYC, ~50ng)	4.3 (pACYC, ~100ng)
DNA insert 1	18 (dxCas9, ~200ng)	17.5 (Tn5Linker, ~200ng)	25 (dxCas9, ~500ng)	39(dxCas9LinkerTn5, ~200ng)	17.5 (Tn5LinkerdxCas9, ~200ng)
DNA nsert 2 (optional)		18 (dxCas9, ~200ng)	5 (LinkerTn5, ~200ng)
MilliQ	0.5	0	0	0	0.2

*L1, L2, L3, L4 and L5 are the names of the ligation reactions. L2 and L3 are single-pot ligation reactions, which aim to ligate two inserts into 1 vector simultaneously.
The samples were incubated for 6 hours at 37°C for ligation. Samples were not heat inactivated.

Date

17/7/2018

Operator

Kavish Kohabir

Solid Medium Preparation

Solid Luria Broth (LB) medium was prepared, complemented with chloramphenicol (Cam)

Date

17/7/2018

Operator

Kavish Kohabir

Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

Strain	Plasmid & size (bp)	Cloning method	Volume added (uL)	Medium
E.coliDH5α	L1 (Ligate)	Restriction&Ligation	20	LB+Cam
E.coliDH5α	L2 (Ligate)	Restriction&Ligation	20	LB+Cam
E.coliDH5α	L3 (Ligate)	Restriction&Ligation	20	LB+Cam
E.coliDH5α	L4 (Ligate)	Restriction&Ligation	20	LB+Cam
E.coliDH5α	L5 (Ligate)	Restriction&Ligation	20	LB+Cam

200 uL LB medium was added as recovery medium.

Date

17/07/2018

Operator

Timothy Páez

Restriction

Restriction cloning was done for assembling Cas9 into pACYCduet lin-Tn5 or lin-Tn5 into pACYCduet dxCas9. Both approaches will render the same insert. The following table shows an overview of what fragments were cut with what restriction enzymes.
First Reaction (lin-Tn5 into pACYC dxCas9):

Component	Volume (uL)
10x CutSmart buffer	2
Fragment (lin-Tn5)	16
Enzyme (Fse HF)	1
Enzyme (KpnI HF)	1
MilliQ	0

Component	Volume (uL)
10x CutSmart buffer	2
Vector (pACYC dxCas9)	16
Enzyme (Fse HF)	1
Enzyme (KpnI HF)	1
MilliQ	0

Second reaction (dxCas9 into pACYC lin-Tn5):

Component	Volume (uL)
10x CutSmart buffer	2
Fragment (dxCas9)	16
Enzyme (NotI HF)	1
Enzyme (FseI HF)	1
MilliQ	0

Component	Volume (uL)
10x CutSmart buffer	2
Vector (pACYC lin-Tn5)	16
Enzyme (NotI HF)	1
Enzyme (FseI HF)	1
MilliQ	0

The samples were incubated for 4 hours at 37 °C.

Date

17/07/2018

Operator

Timothy Páez

PCR clean-up

20 μL of restriction product were subjected to PCR Clean-Up, according to the protocol. Elution was done in 20 µL Milli-Q.

Products were quantified with Nanodrop, yielding concentration of:
- pACYC dxCas9: 42.7 ng/µL
- lin-Tn5: 34 ng/µL
- pACYC lin-Tn5: 31 ng/µL
- dxCas9: 46 ng/µL

Date

17/07/2018

Operator

Timothy Páez

Ligation

In order to assemble cut fragments lin-Tn5 into pACYC dxCas9 (Reaction 1) and dxCas9 into pACYC lin-Tn5, ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

First reaction:

DNA	Size (bp)	Cut with enzymes
pACYCDuet dxCas9	5.5 kbp	Fse and KpnI
lin-Tn5	1.5 kbp	Fse and KpnI

Two different molar ratios between insert and plasmid were tried to attempt higher integration efficiency. Ratios of 1:3 and 1:10 (vector:plasmid) were incubated with T4 ligase (1 µL) and ligase buffer (2 µL). Total volume of reaction was 20 µL.

Second reaction:

DNA	Size (bp)	Cut with enzymes
pACYCDuet lin Tn5	3 kbp	NotI and FseI
dxCas9	4 kbp	NotI and FseI

Two different molar ratios between insert and plasmid were tried to attempt higher integration efficiency. Ratios of 1:3 and 1:10 (vector:plasmid) were incubated with T4 ligase (1 µL) and ligase buffer (2 µL). Total volume of reaction was 20 µL.

The samples were incubated overnight at 4 ºC for ligation.

Date

18/7/2018

Operator

Kavish Kohabir

Transformation Results

After carrying out the transformation protocol on 17/7/2018, the plates were left for overnight incubation. After ~14-18hr incubation, the following results were observed:

Strain	DNA	Medium	Result
E.coli DH5α	L1	LB+Cam	2 colonies
E.coli DH5α	L2	LB+Cam	30 colonies
E.coli DH5α	L3	LB+Cam	2 colonies
E.coli DH5α	L4	LB+Cam	4 colonies
E.coli DH5α	L5	LB+Cam	19 colonies

Date

18/7/2018

Operator

Kavish Kohabir

Colony PCR

Colony PCR was done to verify propagation of ligate constructs after transformation (17/7/2018). This plasmid contains T7 expression system, which can be checked with primers 029 and 030.

Component	L1	L2	L3	L4	L5
Fw primer	029	029	029	029	029
Rv primer	030	030	030	030	030
Expected amplicon size (kbp)	~5.7	~5.7	~5.7	~5.7	~5.7

48 tubes were prepared for colony PCR, containing boiled colony as template DNA. The thermocycler programme was installed to have an extension time of 6.5 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

Date

18/7/2018

Operator

Kavish Kohabir

DNA Gel Electrophoresis

Colony PCR samples were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

It is apparent that no amplicons of interest are found, other than template/uncut/self-closing plasmids (lane 3 & 25). Judging from the double bands around 200 and 500, most of the clones are identified to contain plain pACYC-DUET, which contains double T7 promoter, resulting in double amplicons around that height.

Date

18/07/2018

Operator

Timothy Páez

Transformation of chemically competent cells

Using the ligate reactions from 17/07/2018, the following transformations were carried out according to the protocol for transformation of chemically competent cells:

Strain	Plasmid & size (bp)	Cloning method	Volume added (uL)	Medium on which was plated
E.coliDH5αlpha	pACYC dxCas9-lin-Tn5 (7500 bp)	Restriction Ligation	10 µL	LB with Chloroamphenicol

400 µL LB medium was added as recovery medium.

Date

19/07/2018

Operator

Timothy Páez

Colony PCR

Colony PCR was done to verify propagation of Escherichia coli DH5 alpha cells after transformation with pACYC dxCas9 containing lin-Tn5 (Reaction 1) or pACYC linTn5 containing dxCas9 (Reaction 2) as insert. This insertion can be checked with primers 029 and 030 (on T7 promoter and terminator), but were also tested with primers 001 with 002 for dxCas9 amplification and 005 with 006 for Tn5 amplification. Primer sequences can be found at: Primer sequences.

Component
Template DNA	Colony solution
Forward primer	029, 001, 005
Reverse primer	030, 002, 006
Expected amplicon size (bp)	6 kbp, 4 kbp, 1.5 kbp

The number of colonies screened were:
- Tn5 control : Colony 1
- Cas9-lin-Tn5 (Reaction 1): Colonies 2-10
- Cas9-lin-Tn5 (Reaction 2): Colonies 11-14
The thermocycler programme was installed to have an extension time of 6 min and an annealing temperature of 55 °C. After the programme was finished, samples were kept at 4°C.

Date

19/07/2018

Operator

Timothy Páez

DNA Gel Electrophoresis

Colony PCR products were run on a 0.8 % agarose gel in TAE buffer.
- 5 μL of PCR product was pipetted into each lane (no loading buffer required as PCR buffer contains loading buffer).
- 4 µL of 500 bp DNA ladder
- 80 V for 30 minutes
- The gel was imaged using a gel documentation system with UV-light.

Date

03/08/2018

Operator

Timothy Páez

Liquid Starter Culture Preparation

Strains Escherichia coli DH5 alpha harbouring plasmid with possible fusion protein dxCas9-lin-Tn5 were used for inoculation of 10 mL liquid starter culture (Luria Broth, complemented with chloroamphenicol) following the Liquid Starter Culture Protocol.

Date

04/08/2018

Operator

Timothy Páez

Plasmid Isolation

Overnight starter cultures of Escherichia coli DH5 alpha harbouring plasmid with possible fusion protein dxCas9-lin-Tn5 were subjected to plasmid isolation following the Plasmid Isolation Protocol. Final elution was done with 30 μL of pre-warmed MilliQ.

Date

04/08/2018

Operator

Timothy Páez

High Fidelity PCR

To amplify dxCas9-lin-Tn5 (6 kbp), high fidelity PCR was done using colonies' plasmid harbouring the fusion protein as template DNA and 029 and 030 as forward and reverse primers respectively. Primer sequences can be found at: Primer sequences.

50 µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

The thermocycler programme was installed to have an extension time of 6 minutes and an annealing temperature of 55 °C. After the programme was finished, samples were kept at 4 °C.

Date

03/07/2018

Operator

Timothy Páez

DNA Gel Electrophoresis

PCR products of dxCas9 PCR were run on a 0.8 % agarose gel in TAE buffer.
- 5 μL of PCR product was pipetted into each lane with 1 µL of loading buffer.
- 4 µL of 500 bp DNA ladder
- 80 V for 30 minutes
- The gel was imaged using a gel documentation system with UV-light.

Date

04/08/2018

Operator

Timothy Páez

Sequence Verification

Sequence from plasmid

For verification of dxCas9-Tn5-lin insertion in pACYC, primers 003, 012, 029 and 030 were used for sequencing. Sequencing sample was prepared in 1.5 mL tubes for each primer as indicated my Macrogen for specific concentration of plasmid DNA.
Primer sequences can be found at: Primer sequences.

Within 2 days, the sequence was retrievable online and alignment with the in silico designed molecule showed 100% match of the insert lin-Tn5, by aligning both primer sequences obtained.

Date

08/08/2018

Operator

Nicole Bennis

Liquid Starter Culture Preparation

E.coli strain harbouring the plasmid pACYCDuet-1_Tn5-Lin was used for inoculation of 10 mL liquid starter culture (Luria Broth, complemented with chloramphenicol) following the Liquid Starter Culture Protocol.

Date

09/08/2018

Operator

Nicole Bennis

Plasmid Isolation

Overnight starter culture of pACYCDuet-1_Tn5-Lin were subjected to plasmid isolation following the Plasmid Isolation Protocol. Final elution was done with 30 μL of pre-warmed MilliQ.

Date

09/08/2018

Operator

Nicole Bennis

Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

Isolate	Concentration (ng/uL)	260/280 ratio	260/230 ratio
pACYCDuet-1_Tn5-Lin	66.5	1.88	1.83
pACYCDuet-1_Tn5-Lin	70.1	1.83	1.77
pACYCDuet-1_Tn5-Lin	90.2	1.72	1.06
pACYCDuet-1_Tn5-Lin	108.8	1.73	1.09

Date

09/08/2018

Operator

Nicole Bennis

Restriction

The following table shows an overview of what fragment(s) were cut with what restriction enzyme(s) to obtain fragments Tn5-Lin-dxCas9 and pACYCDuet-1 for ligation.

Component	Volume (uL)	Component	Volume (uL)
10x CutSmart	2	10x CutSmart	2
dxCas9	8.7 (~2000 ng)	pACYCDuet-1_Tn5-Linker	17.2 (~2000 ng)
Enzyme (NotI)	0.4	Enzyme (NotI)	0.4
Enzyme (KpnI)	0.4	Enzyme (KpnI)	0.4
MilliQ	8.5	MilliQ	0

Component	Volume (uL)	Component	Volume (uL)
10x CutSmart	2	10x CutSmart	2
Tn5-Lin	12.5 (~2000 ng)	pACYCDuet-1_dxCas9	17.2 (~2000 ng)
Enzyme (NotI)	0.4	Enzyme (NotI)	0.4
Enzyme (EcoRI)	0.4	Enzyme (EcoRI)	0.4
MilliQ	4.7	MilliQ	0

The samples were incubated for 4 hour(s) at 37 °C, and followed by 20 minute heat inactivation of the restriction enzyme(s).

Date

10/08/2018

Operator

Nicole Bennis

PCR Clean-Up

20 μL of each restriction reaction mixture was subjected to PCR Clean-Up, according to the protocol. Elution was done in 30 µL Milli-Q.

Date

10/08/2018

Operator

Nicole Bennis

Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

Isolate	Concentration (ng/uL)	260/280 ratio	260/230 ratio
Tn5-Lin cut with NotI and EcoRI	88.1	1.83	1.69
pACYCDuet-1_dxCas9 cut with NotI and EcoRI	11.8	1.44	0.69
dxCas9 cut with NotI and EcoRI	34.5	1.76	1.31
pACYC_Tn5-Linker cut with NotI and EcoRI	41.9	1.73	1.36

Date

10/08/2018

Operator

Nicole Bennis

Ligation

In order to assemble cut fragments pACYCDuet-1_Tn5-Linker with dxCas9 Tn-Linker with pACYCDuet-1_dxCas9 respectively, ligation was performed. to optimize the protocol, I used both vector to insert ratio 1:3 and 1:10. This table gives an overview of which fragments were cut with which enzymes.

DNA	Size (bp)	Cut with enzymes
Vector pACYCDuet-1_dxCas9	7929	EcoRI and NotI
Fragment Tn5-Linker	1490	EcoRI and NotI

DNA	Size (bp)	Cut with enzymes
Vector pACYCDuet-1_Tn5-Lin	5258	NotI and KpnI
Fragment dxCas9	4161	NotI and KpnI

A 20 μL ligation reaction mixture was prepared with the following composition:

Component	Volume (μL)	Component	Volume (μL)	Component	Volume (μL)
Ligase Buffer (10X)	2	Ligase Buffer (10X)	2	Ligase Buffer (10X)	2
T4 DNA Ligase	1	T4 DNA Ligase	1	T4 DNA Ligase	1
DNA vector pACYCDuet-1_dxCas9 (~100 ng)	8.5	DNA vector pACYCDuet-1_dxCas9 (~100 ng)	8.5	DNA vector pACYCDuet-1_dxCas9 (~100 ng)	8.5
none		DNA Lin-Tn5 (~60 ng)	0.7	DNA Lin-Tn5 (~190 ng)	2.2
MilliQ	12	MilliQ	11.3	MilliQ	9.8

Component	Volume (μL)	Component	Volume (μL)	Component	Volume (μL)
Ligase Buffer (10X)	2	Ligase Buffer (10X)	2	Ligase Buffer (10X)	2
T4 DNA Ligase	1	T4 DNA Ligase	1	T4 DNA Ligase	1
DNA vector pACYCDuet-1_Tn5-Linker (~100 ng)	2.5	DNA vector pACYCDuet-1_Tn5-Linker (~100 ng)	2.5	DNA vector pACYCDuet-1_Tn5-Linker (~100 ng)	2.5
none		DNA dxCas9 (~220 ng)	5.4	DNA dxCas9 (~730 ng)	14.5
MilliQ	14.5	MilliQ	7.1	MilliQ	0

The samples were incubated for 8 hours at 20°C for ligation.

Date

10/08/2018

Operator

Nicole Bennis

Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

Strain	Plasmid and size (bp)	Cloning method	Volume added (μl)	Medium on which was plated
E.coli DH5α	pACYCDuet-1_dxCas9 restricted (neg control)	Restriction/Ligation	8	LB-Cam
E.coli DH5α	pACYCDuet-1_dxCas9-Linker-Tn5 ratio 1:3 (9449 bp)	Restriction/Ligation	5.5	LB Cam
E.coli DH5α	pACYCDuet-1_dxCas9-Linker-Tn5 ratio 1:10 (9449 bp)	Restriction/Ligation	3	LB Cam
E.coli DH5α	pACYCDuet-1_Tn5-Lin restricted (neg. control)	Restriction	8	LB Cam
E.coli DH5α	pACYCDuet-1_dxCas9-Linker-Tn5 ratio 1:3 (9449 bp)	Restriction/Ligation	3	LB Cam
E.coli DH5α	pACYCDuet-1_dxCas9-Linker-Tn5 ratio 1:10 (9449 bp)	Restriction/Ligation	1	LB Cam

For the transformation 40 ng of total ligate was used. 200 μl LB medium was added as recovery medium.