Difference between revisions of "Team:Pasteur Paris/Notebook"

07.10.2018

Institut Curie’s lab MMBM at IPGG (Institut Pierre Gilles de Gennes) hands us the molds for the microchannel chip and grants us access to their lab. We have been taught the basic steps for the fabrication of the microfluidic microchannel chip: PDMS preparation, molding, demolding, punching, bonding.

07.11.2018

4 microchannel chips have been made. Molds for the PDMS well chip have been received. Glass microscopic slides coated with 2 nm chromium and 50 nm gold have been received for use in PDMS well chip. Glass slides have been cut into small strips with a diamond tip. Processing damaged the gold coating.

07.12.2018

First pieces of the well chip have been successfully molded. 8 microchannel chips have been made.

07.13.2018

Tested the conductivity of gold coated strips of glass. Failure. Too many scratches because of the cutting process. Second successful set of PDMS pieces of the well chip molded. 4 microchannel chips have been made. 1 microchannel successfully bonded to an imaging dish, and filled with fluorescein. Observation under a microscope revealed that it is fully functional.

07.16.2018

We transformed pET 43.1a and pSB1C3 in DH5-α competent cells, in order to constitute a stock of empty vectors for our manipulation.

Plasmid	C (ng/μl)	Volume (μl)	Competent cell	Medium
pET 43.1.a	4.95	1	DH5-α	LB/carbenicilline
pSB1C3	40	1	DH5-α	LB/chloramphenicol

See here the transformation of E. coli DH5-alpha protocol
We let the transformed bacteria grow overnight (16 hours).

07.17.2018

Results:
We went to see our bacterial culture:

• Bacteria transformed with pET 43.1 had not grown.
• Bacteria transformed with pSB1C3 had formed colonies.

Interpretations:
Bacteria transformed with pET 43.1 did not grow. We found a non-commercial tube of pET 43.1 in the freezer from last year team, and we decided to try to amplify it because we did not have any commercial tubes of pET 43.1. Transformation did not work as expected, probably because:

• There was no DNA left in the tube.
• The concentration of DNA was too low .

We cultivated the transformed pSB1C3 bacteria in liquid medium 2 x 25ml + Chloramphenicol (25µg/ml) overnight at 37°C, 180 rpm.

Results:
Bacteria successfully transformed with pSB1C3.
(See here the liquid culture protocol)

07.18.2018

Extraction:
We extracted the pSB1C3 plasmid from the bacterial culture.
The protocol used was the Qiagen Plasmid Purification Kit (See Midiprep for plasmid extraction protocol here )
Measure of the DNA concentration in each tube thanks to the NanoDrop (Blank used : TE.1) (See the NanoDrop protocol here)

Results:
We used the Nanodrop to quantify the purified DNA.

Sample	1	2	3	4	5	6	7	8	9	10
Volume (µl)	50	50	50	50	50	50	50	50	50	50
C (ng/µl)	-7.8	-8.5	54.8	5.6	237.4	-7.9	-7.2	-7.5	-6.9	-6.5
Abs(260 nm)/Abs(280 nm)	1.43	1.42	1.97	3.04	1.85	1.32	1.47	1.56	1.35	1.44

Remarks:
[DNA] < 0 = no DNA
[DNA] > 200 good
Abs_{(260 nm)}/Abs_{(280 nm) [1;2]}

Interpretations:

• Tube 1, 2, 6, 7, 8, 9 and 10 did not contain DNA at all. This problem can be due to the fact that we failed the precipitation with ethanol, meaning that when we removed all the supernatant, we also removed the DNA, or that we removed by mistake the DNA pellet when removing all the supernatant after precipitation (the pellet was so small that we couldn’t see it with the naked eye).
• Tube 3, 4 and 5 had DNA inside. The amount of DNA obtained is enough for us. These tubes have been placed in the freezer for later use.

Transformation:
We received from Eurofins our first sequences:

• 3a_NGF construction Part1 (Seq1)
• 3a_NGF construction Part2 (Seq2)
• T7 RIP construction (Seq8)

In order to constitute a stock of this commercial vectors, we transformed DH5-α competent cells with the plasmid we received, and then we cultivated them on agar plates covered with LB medium and carbenicillin.
As the bacteria transformed with pET 43.1a did not grow, we tried again to transform the bacteria with pET 43.1a in a higher quantity than previously.

Plasmid	C (ng/μl)	Volume (μl)	Competent cell	Medium
pEX-A258 Seq1	10	1	DH5-α	SOC/carbenicilline
pEX-A258 Seq2	10	1	DH5-α	SOC/carbenicilline
pEX-A258 Seq8	10	1	DH5-α	SOC/carbenicilline
pET 43.1a	4.95	5	DH5-α	SOC/carbenicilline

See here the transformation of E. coli DH5-alpha protocol

07.19.2018

Results:
We went to see our overnight bacterial culture plates:

	SEQ#1 3a_NGF_Part1	SEQ#2 3a_NGF_Part2	SEQ#8 T7_RIP	Pet43.1a(+)
Selection	Amp	Amp	Amp	Amp
Growth	Yes	Yes	Yes	No
Liquid Culture	Yes	Yes	Yes	No

Interpretations:
Transformation of Seq#1 – 3a_NGF_Part1, Seq#2 – 3a_NGF_Part2 and Seq #8 – T7_RIP did work as expected.
Transformation of Pet43.1a (+) did not work again. As mentioned before, the tube of pET43.1a (+) was a non-commercial tube that we found in the freezer from last year’s team, and we decided to try to amplify it because we did not have any commercial tubes of pET 43.1. We tried to transform our bacteria twice, with no results, so the most probable hypothesis is that the tube did not contain DNA anymore. After this experiment, we decided to buy a tube pET43.1a(+).

We cultivated two different colonies from each plate (seq#1, seq#2, seq#8):

• One in an Erlenmeyer in 25 ml LB medium + Carbenicillin (100µg/ml) overnight at 37°C, 180 rpm.
• One in two Falcon tube in 15 ml LB medium + Carbenicillin (100µg/ml) overnight at 37°C, 180 rpm.

(See here the liquid culture protocol)

07.20.2018

Results of the liquid culture are good.

Extraction - MidiPrep:
We extracted the 3 plasmids Seq#1 – 3a_NGF_Part1, Seq#2 – 3a_NGF_Part2 and Seq #8 – T7_RIP from the bacterial cultures.
The protocol used was the Qiagen Plasmid Purification Kit (See Midiprep for plasmid extraction protocol here )
Measure of the DNA concentration in each tube thanks to the NanoDrop (Blank used : TE.1) (See the NanoDrop protocol here)

Results:
We used the Nanodrop to quantify the purified DNA.

For NGF part1, Colony 1:

ng/µL [DNA]	Abs(260 nm)/Abs(280 nm)
3569.2	1.9
1578.7	1.89
848.1	1.90
2322.1	1.89

For NGF part1, Colony 2:

ng/µL [DNA]	Abs(260 nm)/Abs(280 nm)
488.1	1.89
493.7	1.90
445.1	1.89
644.6	1.86

For NGF part2, Colony 1:

ng/µL [DNA]	Abs(260 nm)/Abs(280 nm)
1555.0	1.88
493.7	1.88
666.2	1.87
367	1.90

For NGF part2, Colony 2:

ng/µL [DNA]	Abs(260 nm)/Abs(280 nm)
463.7	1.89
420.8	1.88
461.4	1.89
420.6	1.89

For RIP, Colony 1:

ng/µL [DNA]	Abs(260 nm)/Abs(280 nm)
694.4	1.84
1028.9	1.87
1408.7	1.88
466.8	1.88

For RIP, Colony 2:

ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
228.4	1.89
325.2	1.89
379.4	1.89
427.2	1.89

Remarks:
[NA] < 0 = no DNA
[NA] > 200 good
Abs_{(260 nm)}/Abs_{(280 nm) [1;2]}

Interpretations:
We have DNA in our tubes. The DNA is too concentrated so we will have to dilute them before next use. We froze the tubes at -20°C until next use.

Something was done one that day

18.07.2018

1 set of PDMS pieces for the well chip has been made. One of the pieces broke while demolding. It was discarded. 4 microchannel chips made.

19.07.2018

1 set of PDMS pieces for the well chip have been made. 4 microchannel chips made.

Something was done one that day

07.24.2018

Culture of strain HB2151 transformed with pVDL 9.3 (received on 23.07.2018 from Dr. Victor de Lorenzo)

Material:

• LB agar plate supplemented with chloramphenicol (25 mg/mL)
• Bacteria HB 2151 transformed with pVDL9.3

The bacteria we received was in the form of a stock: LB + agar media containing the bacteria and the plasmid in an Eppendorf tube. Sample from the solid stock was streaked on LB agar plate complemented with chloramphenicol, and secondly grown in an Erlenmeyer with 25 mL LB medium and 25 μL chloramphenicol at 25 mg/mL.

Biofilm culture on filter (0.2 μm)

Material:

• Bacteria DH5α transformed with pSB1C3
• Filters - 0.2 μm Milipore (5 cm diameter)
• Petri-dishes
• Sterile Clamps
• Sterile Rakes
• Pipets
• LB media
• Falcon tubes
• Chloramphenicol (25 mg/mL)

1. Make a liquid culture of DH5α transformed with pSB1C3 from colonies we had on a petri dish made on the 07.16.2017 and stored at 4°C. Let grow over night at 37°C under agitation.
   Made the 07.23.2017
   (See here the liquid culture protocol)
2. Auto-cleavage of the 0.2 μm filters
3. Deposit the filters on a petri dish with a clamp. Wait until the filter is totally “wet” with LB media.
4. Deposit the bacteria culture in the center of the filter with a micropipette and spread with a rake. We made 3 different petri dishes with 3 different volumes.

	DH5α-pSB1C3- biofilm culture – 1	DH5α-pSB1C3- biofilm culture – 2	DH5α-pSB1C3- biofilm culture – 3
Volume of culture deposit (μL)	50	100	150

5. Incubate at 37°C overnight.

Transformation of seq5, seq7 from Eurofins Genomics, pET 43.1 and pBR322
Material:

• LB agar plate supplemented with carbenicillin
• Sterile LB medium
• pET 43.1a: 42.36 pg/μL
• pBR322: 65.00 ng/μL
• Seq5 > AGR-RIP Part1: 1.1 μg
• Seq7 > AGR-RIP Part3: 1.9 μg

Sequences received on the 07.24.2018.
Gene synthesis products are in the form of lyophilized powder, so we need to re-suspend them.

Seq5 > AGR-RIP Part1: 1.1 μg
Seq7 > AGR-RIP Part3: 1.9 μg

1. Centrifuge in order to make sure that all the plasmids are at the bottom of the tube and not in the lid.
2. Re-suspend the powder in TE 1X.
   Seq5: add 110 μL to have a final concentration of 10 ng/μL
   Seq7: add 190 μL to have a final concentration of 10 ng/μL
3. Transformation of the 4 plasmids in DH5α competent cells:
   • Seq5, Seq7 and pBR322: 1 μL
   • pET 43.1a: 10 μL
   
   (See here the transformation of E. coli DH5-alpha protocol)
4. Culture on petri dishes overnight at 37°C

07.25.2018

Transformed bacteria cultures:
We went to see our overnight bacterial culture plates:

	Seq5 RIP_Part1	Seq7 RIP_Part3	pBR322	Pet43.1a(+)
Selection	Amp	Amp	Amp	Amp
Growth	Yes	Yes	Yes	No
CFU	2 816	2 492	2 628	-
Liquid Culture	Yes	Yes	Yes	No

Interpretations:
Transformation of Seq5 – RIP_Part1, Seq7 – RIP_Part3 and pBR322 did work as expected.
Transformation of Pet43.1a (+) did not work again. The tube of pET43.1a (+) was another non-commercial tube that we found in the freezer from last year’s team, and we decided to try to amplify it because we did not have any commercial tubes of pET 43.1. We tried to transform our bacteria twice, with no results, so the most probable hypothesis is that the tube did not contain DNA anymore.
We are still waiting for the commercial pET43.1a(+) (we ordered it last week).

Bacteria transformed pVDL 9.3:
The culture on agar plate did work, but we were not able to isolate one colony.
We stopped the liquid culture and measured the OD600 of the culture medium.

Biofilm culture:

	pSB1C3-DH5α-biofilm culture – 1	pSB1C3-DH5α-biofilm culture – 2	pSB1C3-DH5α-biofilm culture – 3
Volume of culture deposit (μL)	50	100	150
Biofilm formation	Yes	Yes	Yes

We did a biofilm transfer. We took our filter with the biofilm and transferred it on another porous filter placed on a new petri dish LB + Cm. Then we removed the filter containing the biofilm and incubated the plate overnight at 37°C.

Liquid culture:
We cultivated two different colonies from each plate (Seq5, Seq7 and pBR322):

• One in an Erlenmeyer in 25 mL LB medium + Carbenicillin (100 µg/ml) overnight at 37°C, 180 rpm.
• One in two Falcon tube in 15 mL LB medium + Carbenicillin (100 µg/ml) overnight at 37°C, 180 rpm.

Moreover, for each colony of each DNA part we did some streaks on a plate and incubated them at 37°C. (See here the liquid culture protocol)

Midiprep pVDL 9.3:
We extracted the pVDL 9.3 plasmid from the HB2151 strain.
The protocol use was the Qiagen Plasmid Purification Kit (See Midiprep for plasmid extraction protocol here )
We divided the eluted DNA into 8 tubes before centrifugation.

Measure of the DNA concentration in each tube by the NanoDrop.
Blank used : TE.1 (See the NanoDrop protocol here)

Results:
We used the Nanodrop to quantify the purified DNA for SEQ: pVDL 9.3 1.

Sample	1	2	3	4	5	6	7	8
ng/µL [NA]	3.7	111.2	140	0	1.9	47	108	8.3
Abs(260 nm)/Abs(280 nm)	1.93	1.87	1.88	-	1.6	1.9	1.9	1.91

07.26.2018

Results:
Transfer of our biofilm growth on a filter (07.24.2018 loading of 50 µl of pSB1C3 liquid culture on a filter) on another filter on a plate and incubation overnight at 37°C:

Observations:
The biofilm is able to grow again after a transfer from one filter to another as seen by the red color.

Midiprep Seq5, Seq7, pBR322:
We extracted Seq5, Seq7 and pBR322 plasmid from transformed DH5α strain.
The protocol used was the Qiagen Plasmid Purification Kit (See Midiprep for plasmid extraction protocol here )
We divided the eluted DNA into 4 tubes before centrifugation.

Measure of the DNA concentration in each tube with NanoDrop.
Blank used: TE.1 (See the NanoDrop protocol here)

Results:
We used the Nanodrop to quantify the purified DNA.

For Seq, 5 Colony 1:

ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
245.7	1.87
349	1.87
353.2	1.87
306.7	1.87

For Seq 5, Colony 2:

ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
258	1.88
281	1.86
260	1.86
295	1.86

For Seq 7, Colony 1:

ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
293	1.88
299	1.88
305	1.87
288	1.87

For Seq 7, Colony 2:

ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
217	1.87
212	1.87
175	1.88
244	1.83

For pBR322, Colony 1:

ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
3.8	1.68
55.8	1.86
54	1.9
68	1.84

For RIP, Colony 2:

ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
32	1.82
28	1.75
41	1.88
30	1.88

Interpretations:
There is DNA in all the tubes. For Seq5 and Seq7, we obtain high DNA concentrations, which is good. For pBR322, the DNA concentration is lower. This is probably due to the fact that pB322 is a low copy plasmid. However, the quantity of DNA is enough.

Preparing cultures for stocks:
We set some pre-cultures of Seq5, Seq7 and pBR322, in order to make some stocks to store at -80°C. In a 15 mL Falcon tube, we placed 5 mL of LB media, and 10 μL of antibiotic (Carbenicillin). We added one of each colony inside each Falcon and placed the Falcon tubes inside the incubator (37°C – 180 rpm) and let grow overnight.

Something was done one that day

07.24.2018

Membrane filters from Sterlitechreceived. 8 microchannel chips and 1 set of PDMS pieces for the well chip have been made.

07.25.2018

Platinum wires received and conductivity tested. Success.

07.27.2018

4 microchannel chips have been made. 1 microchannel chip bonded for imaging test purposes.

Something was done one that day

07.30.2018

Digestion of Seq8 and pBR322:
We cut the commercial plasmid containing our Seq8 (T7-RIP) with restriction enzymes XbaI and SpeI, and pBR322 with AvaI and BsmI, in order then to insert our sequence into the plasmid pBR322.
Material:

• Water baths at 37°C and 80°C
• Eppendorf tubes
• Pipets and cones

Reagents:

• pBR322 [68 ng/μL]
• commercial plasmid containing Seq8 [466,8 ng/μL]
• Restriction enzymes: AvaI, BsmI, XbaI, SpeI
• 10X Cut Smart
• Distilled water

1. Mix the following volumes of reagents and incubate at 37°C for 15 minutes.

	Volume (μL)
pBR322	22
10X Cut Smart	5
Distilled water	20
Bsml	1.5
Aval	1.5

	Volume (μL)
Eurofins plasmid with Seq8	20
10X Cut Smart	5
Distilled water	22
Xbal	1.5
Spel	1.5

2. Inactivate the digestion by heating the mixture in a heat block at 80 °C for 20 minutes.

 

Electrophoresis on agarose gel

We prepared an Agarose gel.
(See here the Agarose Gel Preparation protocol)

We filled:

• 1 well with the digested pBR322 (n°1)
• 2 wells with the digested plasmids containing Seq8 (n°2 and 3)
• 1 well with a DNA ladder (n°4)

We let the DNA migrate for 10 minutes at 85 V and then 40 minutes at 125 V.
We then revealed the gel under UV lamp.

Results:

Interpretations:

• pBR322 (4361 bp) has been successfully linearized
• pEXA258 from Eurofins Genomics has also been successfully digested. We obtained two fragments corresponding to our insert (459 bp) and the linearized plasmid (2909 bp)

DNA Gel extraction:
Here is the same gel after DNA extraction of pBR322 and of Seq8.

Sequence name	Eppendorf weight (g)	Eppendorf + gel weights (g)	Gel weight (g)
Seq8 (1)	1.08	1.29	0.21
Seq8 (2)	1.08	1.37	0.29
pBR322	1.08	1.27	0.19

It is useful to know how the weight of the gel to adapt de volumes of buffer to the amount of gel we have.
(See here the Gel Extraction protocol)
Results: after extracting the DNA from the gel, we measured the DNA concentration using the Nanodrop.

Sequence name	DNA concentration (ng/μL))	Abs(260nm)/Abs(280nm) (g)	Total extracted DNA (ng)
Seq8 (1)	6.4	1.87	294.4 (620.8)
Seq8 (2)	6.8	2.07	326.4 (620.8)
Linearized pBR322	9.9	1.97	475.2

Interpretations:
The initial amount of each DNA fragment before digestion was 1500 ng.
We can therefore evaluate the efficiency of our digestion and extraction:

• for Seq8 insert: r = 0.414
• for pBR322 plasmid: r = 0.317

Ligation of linearized pBR322 and our insert Seq8

We used the In-Fusion cloning kit provided by Ozyme in order to clone our insert into the linearized pBR322.

1. We mixed the following volumes into 1.5 mL tubes.

Component	Cloning (μL)	Negative control (μL)	Positive control (μL)
Purified fragment	4	-	2 (control)
Linearized vector	4	1	1 (control)
5X In-Fusion enzyme premix	2	2	2
Deionized water	-	7	5

2. Incubate for 15 min in a water bath at 50°C
3. Ligation See here the Ligation protocol)
4. Set in ice
5. Transformation of the 3 plasmids into Stellar competent cells (provided with the cloning kit)
   • Competent cell volume : 33 μL
   • DNA volume: 2 μL
   (See here the transformation of E. coli DH5-alpha protocol)
6. Culture on petri dishes LB agar gel supplemented with carbenicillin overnight at 37°C

Transformation of Seq6 from Eurofins
Material:

• LB agar plates supplemented with carbenicillin (25 μg/mL)
• LB medium
• Seq6 > AGR-RIP Part1: 4.6 μg

Sequences are under the form of lyophilized powder, so we need to re-suspend it.

1. Centrifuge in order to make sure that all the plasmids are at the bottom of the tube and not in the lid.
2. Re-suspend the powder in TE 1X.
   We resuspended it into 460 μL TE, to obtain a concentration of 10 ng/μL.
3. Transformation of the plasmid in DH5α competent cells
   (See here the transformation of E. coli DH5-alpha protocol)
4. Culture on petri dishes LB agar gel supplemented with carbenicillin overnight at 37°C

07.31.2018

Results: ligation cultures

As expected, we could observe a lot of colonies for the positive control. Some colonies had grown for the negative control (due to the linearized plasmid which circularized itself). For our ligated plasmid, we only observed a few colony (as much as in the negative control) so we didn’t know if the ligation succeeded. This is why we picked up 20 colonies and cultivated them overnight, hoping that one of these had the correct plasmid.

The plate containing DH5-α transformed with Seq6 also had a lot of colonies, as expected.

Liquid culture of DH5-α with Seq6
We cultivated 2 different colonies in 250 mL Erlenmeyer containing:

• 25 mL LB medium
• 25 μL carbenicillin (25 mg/mL)

We cultivated it overnight in a shaking incubator at 37°C, 180 rpm.
(See here the liquid culture protocol)

Liquid culture of Stellar transformed with pBR322-Seq8

We cultivated 20 different colonies in order to maximize our chance to get one successful ligated plasmid. Each colony was cultivated in a 15 mL Falcon tube containing:

• 5 mL LB medium
• 5 μL carbenicillin (25 mg/mL)

We cultivated it overnight in a shaking incubator at 37°C, 180 rpm.
We also cultivated each colony on petri dishes LB agar plate supplemented with carbenicillin overnight at 37°C.

Transformation of Seq3 from Eurofins Genomics and commercial pET43.1a
Material:

• LB agar plate supplemented with carbenicillin
• Seq3 : NGF-GFP Part1 : 4.0 μg
• pET 43.1a : 0.5 μg/μL

Sequence from Eurofins Genomics under the form of lyophilized powder so we need to re-suspend it.

1. Centrifuge in order to make sure that all the plasmids are at the bottom of the tube and not in the lid.
2. Re-suspend the powder in TE 1X. We resuspended it into 400 μL TE, in order to obtain a concentration of 10 ng/μL.
3. Transformation of the plasmids in DH5α competent cells:
   • volume of competent cells: 50 μL
   • pET 43.1a: 50 pg (1 μL of commercial plasmid diluted 1/10000)
   • Seq3 : 10 ng (1 μL)
   (See here the transformation of E. coli DH5-alpha protocol)
4. Culture on petri dishes LB agar plate supplemented with carbenicillin overnight at 37°C.

08.01.2018

We went to see our cultures:

• All the colonies of our ligation (pBR322 + Seq8) grew except one.
• DH5α transformed with Seq3 grew.
• DH5α transformed with pET 43.1a did not grow.

Hypothesis:

• We might have diluted the plasmid or the bacteria too much.
• We did not homogenize the solution enough during the dilution of the commercial solution.
• We did not pipet correctly the 1 μL of plasmid from the commercial tube.

 

Extraction of the ligated plasmid (pBR322 + Seq8) from Stellar (miniprep)
(See here the Gel Extraction protocol)
Results: (tube 8 did not grow)
From NanoDrop

Sample	DNA CONCENTRATION (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	94.8	2.05
2	105.8	2.06
3	106.3	2.04
4	84.8	2.02
5	100.1	2.02
6	79.9	2.00
7	08.9	2.04
8	-	-
9	115.1	2.02
10	116.7	1.96
11	76.6	2.00
12	69.5	1.99
13	80.7	1.94
14	63.0	1.98
15	78.4	2.03
16	153.1	1.94
17	90.7	2.00
18	119.3	2.02
19	100.8	1.97
20	85.3	2.03

We chose to verify which colony had acquired the plasmid containing the Seq8 insert. To do so, we digested the plasmid with the restriction enzymes EcoRI and PstI and ran a 1% agarose gel.
The size of the plasmid pBR322 is 4361 bp. Once we cut the sequence between the sites EcoRI (bp position 4359) and PstI (bp position 3611), this leaves us with an empty backbone of 3613 bp. The size of the insert Seq8 is 461 bp.
Here we made a mistake, by confusing the right restriction enzymes to use. Indeed, we should have used XbaI and SpeI instead of EcoRI and PstI. Moreover, as these two restriction sites are present in the pBR322 backbone (748 bp away), this digestion should show two DNA fragments.

1% Agarose gel of the miniprep of the ligated plasmid (pBR322 + Seq8) from Stellar
Only the first half of the samples (1 to 11) were deposed today.
The preparation of the gel was done according to the Agarose Gel Preparation protocol.
The samples were made according to the following proportions:

• 5 µL DNA
• 3 µL 10X Cutsmart Buffer
• 1 µL EcoRI
• 1 µL PstI
• 20 µL H2O

These samples were then incubated for 10 min at 37°C to allow digestion by the enzymes.
The reaction was stopped by the addition of 6 µL of 6X Loading Dye.
Results:

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This resulting gel shows the two DNA fragments we expected. Moreover, the column 3 shows a lower molecular weight (MW) than others for the plasmid part. As this part is supposed to contain our insert, it might suggest that colony 3 doesn’t possess the insert, but all others do.
However, we will need to make the right experiment using restriction enzymes XbaI and SpeI (see date 08.02.2018) to verify that our insert was correctly put into the plasmid.

Extraction of Seq6 plasmid from DH5α (midiprep)

We extracted the plasmids containing Seq6 from the DH5-α cells.
(See Midiprep for plasmid extraction protocol here )

Results from NanoDrop: (Blank: TE.1)

For the colony 1:

	ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
1	1572.9	1.88
2	2800.1	1.88
3	2598.7	1.88
4	2285.1	1.89

For the colony 2:

	ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
1	4996.8	1.87
2	3263.2	1.89
3	3625.0	1.90
4	2339.7	1.90

The DNA concentrations were too high and so did not answer to the Beer-Lambert’s law. We needed to dilute the samples before using the nanodrop again. We did 1/10 dilutions and obtained average concentrations of 34 ng/µL, with Abs_{(260 nm)}/Abs_{(280 nm)} ratios of 1.92.

Digestion of pET 43.1a, NGF_Part1 (Seq1) and NGF_Part2 (Seq2)

Digestion of the following plasmids:

• pET43.1a (500 ng/µl)
• Commercial plasmid containing Seq1 (445.1 ng/μL)
• Commercial plasmid containing Seq2 (420.6 ng/μL)
• Restriction enzymes: NdeI, PacI, NheI, BamHI, MscI, HindIII

1. Mix the following volumes of reagents and incubate at 37°C for 15 min.

	Volume (μL)
pET43.1a	1.6
10X Cut Smart	5
Distilled water	40.4
Ndell	1.5
Pacl	1.5

	Volume (μL)
Eurofins plasmid with Seq1	4.3
10X Cut Smart	5
Distilled water	37.7
Nhel	1.5
BamHI	1.5

	Volume (μL)
Eurofins plasmid with Seq2	4.4
10X Cut Smart	5
Distilled water	37.6
Mscl	1.5
MindIII	1.5

2. Inactivate the digestion by heating the mixture in a heat block at 80 °C for 20 min.

Electrophoresis on agarose gel

We prepared an 0.8 % agarose gel to separate the different fragments of the cut plasmids.
(See here the Agarose Gel Preparation protocol)

We filled:

• 2 wells with digested pET43.1a (n°1 and n°2)
• 2 wells with insert Seq1 (n°3 and n°4)
• 2 wells with insert Seq2 (n°5 and n°6)
• 1 well with the DNA ladder

We let the DNA migrate for 10 minutes at 85 V on order to let the DNA penetrate the gel, and then 40 min at 125 V to efficiently separate the fragments.
We then revealed the gel under UV lamp.

Interpretations:

• pET43.1a (7275 bp) has been successfully linearized into two fragments (5 kb and 2 kb)
• pEXA258 – Seq1 from Eurofins Genomics has also been successfully digested. We obtained two fragments corresponding to our insert (1105 bp) and the linearized plasmid (2446 bp)
• pEXA258 – Seq2 from Eurofins Genomics has also been successfully digested. We obtained two fragments corresponding to our insert (1159 bp) and the linearized plasmid (2446 bp)

DNA Gel extraction:
We cut the gel to extract our inserts and linearized plasmid:

	Eppendorf weight (g)	Eppendorf + gel weights (g)	Gel weight (g)
pET43.1a (1)	1.13	1.365	0.23
pET43.1a (2)	1.08	1.286	0.21
Seq1 (1)	1.07	1.265	0.20
Seq1 (2)	1.08	1.306	0.23
Seq2 (1)	1.07	1.303	0.23
Seq2 (2)	1.07	1.284	0.21

It is useful to know how the weight of the gel to adapt de volumes of buffer to the amount of gel we have.
(See here the Gel Extraction protocol)

Results: After extracting the DNA from the gel, we measured the DNA concentration using the Nanodrop.

	C (ng/μL)	Abs(260 nm)/Abs(280 nm)
pET43.1a (+) (1)	5.5	3.14
pET43.1a (+) (2))	7.1	2.50
Seq1 (1)	5.1	2.71
Seq1 (2)	7.8	1.90
Seq2 (1)	6.9	2.92
Seq2 (2)	6.0	3.11

Ligation of linearized pET43.1a and our inserts Seq1,2 /br>
We used the In-Fusion cloning kit provided by Ozyme to clone our two inserts together in linearized pET43.1a.

1. We mixed the following volumes into 1.5 mL tubes.

Component	Cloning (μL)	Negative control (μL)	Positive control (μL)
Purified Seq1	6	-	2 (control)
Purified Seq2	6	-	2 (control)
Linearized vector	3	1	1 (control)
5X In-Fusion enzyme premix	4	2	2
Deionized water	1	7	5

2. Incubate for 15 min in a water bath at 50°C
3. Ligation (See Ligation protocol)
4. Set on ice
5. Transformation of the 3 plasmids into Stellar competent cells (provided with the cloning kit):
   • Competent cell volume: 50 μL
   • DNA volume: 2 μL
   (See here the transformation of E. coli DH5-alpha protocol)


6. Culture on petri dishes LB agar plate supplemented with carbenicillin overnight at 37°C

08.02.2018

2nd Agarose gel of the miniprep of the ligated plasmid (pBR322 + Seq8) from Stellar, using the correct enzymes
Yesterday we made a mistake, so today we carried out exactly the same protocol but with restriction enzymes SpeI and XbaI. As a negative control, we also deposited a sample we called 1x, without digesting it with restriction enzymes.

Liquid culture of pET43 1.a
We cultivated 2 different colonies in 250 mL Erlenmeyer containing:

• 25 mL LB medium
• 25 μL carbenicillin (25 mg/mL)

We cultivated it overnight in a shaking incubator at 37°C, 180 rpm.
(See here Liquid Culture protocol)

Liquid culture of Stellar transformed with pET43 1.a-Seq1-Seq2
We cultivated 20 different colonies in order to maximize our chance to get one successful ligated plasmid. Each colony was cultivated in a 15 mL Falcon tube containing:

• 5 mL LB medium
• 5 μL carbenicillin (25 mg/mL)

We cultivated it overnight in a shaking incubator at 37°C, 180 rpm.
< We also cultivated each colony in an LB agar plate supplemented with carbenicillin.

Extraction of Seq3 plasmid from Stellar (midiprep)
We extracted the plasmids containing Seq3 from the stellar cells
(See here the Midiprep for plasmid extraction protocol)

Results from NanoDrop (with Blank: TE.1)
For the colony 1:

ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
153.3	1.90
155.2	1.90
164.6	1.89
179.1	1.91

For the colony 2:

ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
166.4	1.91
150.4	1.90
177.1	1.91
188.6	1.89

Interpretations: There is DNA in all the tubes

Digestion of pBR322, Agr-RIP-Part1 (Seq5), Agr-RIP-Part2 (Seq6) and Agr-RIP-Part3 (Seq7)

Material:

• Water baths at 37°C and heating block of 80°C
• Eppendorf tubes
• Pipets and cones

Reagents:

• PBR322 (conc. 68 ng/μL)
• Eurofins Genomics plasmid containing Seq5 (conc. 353 ng/ μL)
• Eurofins Genomics plasmid containing Seq6 (conc. 350 ng/ μL)
• Eurofins Genomics plasmid containing Seq7 (conc. 305 ng/ μL)
• Restriction enzymes: BsmI, AvaI, XbaI, BamHI, MscI, SphI, HindIII, SpeI
• 10X Cut Smart
• Distilled water

1. Mix the following volumes of reagents and incubate at 37°C for 15 min

	Volume (μL)
pBR322	122
10X Cut Smart	5
Distilled water	20
BsmI	1.5
Aval	1.5

	Volume (μL)
Plasmid containing Seq5	12
10X Cut Smart	5
Distilled water	30
Xbal	1.5
BamHI	1.5

	Volume (μL)
Plasmid containing Seq7	21
10X Cut Smart	5
Distilled water	21
HindIIIl	1.5
Spel	1.5

	Volume (μL)
Plasmid	16
10X Cut Smart	5
Distilled water	26
Mscl	1.5
Sphl	1.5

2. Inactivate the digestion by heating the mixture in a heat block at 80 °C for 20 min.

Electrophoresis on agarose gel

We prepared an 0.8 % agarose gel to separate the different fragments of the cut plasmids.
(See here the Agarose Gel Preparation protocol)

We filled:

• 2 wells with digested pBR322 (n°1 and n°2)
• 2 wells with insert Seq5 (n°3 and n°4)
• 2 wells with insert Seq6 (n°5 and n°6)
• 2 wells with insert Seq7 (n°7 and n°8)
• 1 well with the DNA ladder

We let the DNA migrate for 10 minutes at 85 V on order to let the DNA penetrate the gel, and then 40 min at 125 V to efficiently separate the fragments.
We then revealed the gel under UV lamp.

• 1. 2. : pBR322
• 3. 4. : Seq5
• 5. 6. : Seq6
• 7. 8. : Seq7

Interpretations:

• pET43.1a (7275 bp) has been successfully linearized into two fragments
• pEXA258 – Seq5 from Eurofins Genomics has also been successfully digested. We obtained two fragments corresponding to our insert (1422 bp) and the linearized plasmid (2.5 kb)
• pEXA258 – Seq6 from Eurofins Genomics has also been successfully digested. We obtained two fragments corresponding to our insert (960 bp) and the linearized plasmid (2.5 kb)
• pEXA258 – Seq7 from Eurofins Genomics has also been successfully digested. We obtained two fragments corresponding to our insert (762 bp) and the linearized plasmid (2.5 kb)

DNA Gel extraction
We cut the gel to extract our inserts and linearized plasmid:








We extract the DNA (our linearized vector and our plasmid) from the agarose gel.
(See here the Gel Extraction protocol)



Results: After extracting the DNA from the gel, we measured the DNA concentration using the Nanodrop

	ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
pBR322	10.3	2.07
Seq5	8.3	3.07
Seq6	14.0	2.06
Seq7	7.6	1.97




Ligation of linearized pBR322 and our insert Seq5,6,7

We used the In-Fusion cloning kit provided by Ozyme (See here theLigation protocol) in order to clone our insert into the linearized pBR322.


1. We mixed the following volumes into 1.5 mL tubes.

Component	Cloning (μL)	Negative control (μL)	Positive control (μL)
Purified Seq 5	5	-	2 (control)
Purified Seq 6	3	-	2 (control)
Purified Seq 7	5	-	2 (control)
Linearized vector	3	1	1 (control)
5X In-Fusion enzyme premix	4	2	2
Deionized water	-	7	5







2. Incubate for 15 min in a water bath at 50°C
3. Ligation See here the Ligation protocol)
4. Set in ice
5. Transformation of the 3 plasmids into Stellar competent cells (provided with the cloning kit)
   • Competent cell volume : 33 μL
   • DNA volume: 2 μL
   (See here the transformation of E. coli DH5-alpha protocol)
6. Culture on petri dishes LB agar gel supplemented with carbenicillin overnight at 37°C




08.03.2018





Midiprep pET43.1a:
(See here the Midiprep for plasmid extraction protocol)

2 colonies from pET43.1a petri dish (08.01.2018 – pET43.1a) were placed in liquid culture the day before

Results from NanoDrop (with Blank: TE.1)
For the colony 1:


ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
71.7	1.88
75.2	1.88
73.6	1.84
70.2	1.87

For the colony 2:


ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
65.0	1.85
34.7	1.80
64.9	1.86
55.5	1.82




Observations: Enough concentrations for further uses






Miniprep pET 43.1a + Seq1 + Seq2
(See here the Miniprep for plasmid extraction protocol)
20 colonies from pET43.1a + Seq1 + Seq2 from petri dish (08.01.2018) were placed in liquid culture the day before (5 mL of LB + 10 µL of Carbenicillin).

Results from NanoDrop (Blank used is Buffer EB):

Sample	DNA CONCENTRATION (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	170.9	1.86
2	120.8	1.88
3	73.1	1.87
4	89.8	1.84
5	75.4	1.97
6	136.6	1.87
7	101.4	1.88
8	99.0	1.91
9	72.7	1.88
10	172.8	1.87
11	107.8	1.86
12	151.1	1.86
13	92.6	1.92
14	78.7	1.93
15	101.2	1.87
16	223.9	1.97
17	145.5	1.85
18	149.8	1.86
19	68.1	1.89
20	87.8	1.93




Observations: Samples were not transferred to the micro tubes at the right moment, so it is very probable that the samples are contaminated with genomic DNA.





08.05.2018





Liquid culture of pBR322 + Seq5 + Seq6 + Seq7
(See here the Liquid Culture protocol)
6 colonies from the petri dish (pBR322 + Seq5,6,7 – 08.02.2018) were placed in a liquid culture containing:

• 5 mL of LB
• 10 µL of Carbenicillin (25 mg/mL)


Cultures were incubated at 37°C, 180 rpm overnight.

Observations: All the colonies grew.

Something was done one that day

07.30.2018

First PDMS well chip entirely assembled and bonded, with platinum wire and gold membrane filter. Leaking problems required us to seal the leaks with additional PDMS.4 microchannel chips and 1 set of PDMS pieces for the well chip have been made.

07.31.2018

2 additional PDMS wells assembled. First PDMS vertical chip PDMS pieces prepared (cut and punched, silver paste applied and membrane filter glued). 4 microchannel chips and 1 set of PDMS pieces for the well chip have been made.

08.01.2018

Conductivity tests on membranes assembled on chips. Well chips not functional. It was decided for the well chips to stop using silver paste for connecting the platinum wire to the membrane and instead mount the wire on top of the membrane. Bonding of the vertical chip PDMS pieces failed

Something was done one that day

08.06.2018

Miniprep pBR322 + Seq5 + Seq6 + Seq7
(See here the Miniprep for plasmid extraction protocol)

Results from NanoDrop (Blank used is buffer EB):

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	61.2	2.00
2	86.3	2.03
3	58.8	1.98
4	65.9	2.00
5	74.5	2.03
6	84.4	2.07

Observations: Enough concentrations for further use.

Digestion of pET43.1a + Seq1 + Seq2

Aim: Check that the ligation has successfully worked.
Restriction enzymes used: NdeI and XbaI

Expected results:

pET43.1a	linearized	7.3 kB
pET43.1a + Seq1	1 fragment	6.63 kB
pET43.1a + Seq2	2 fragments	6.63 kB + 0.1 kB
pET43.1a + Seq1+ Seq2	2 fragments	6.63 kB + 1 kB

We digested 10 samples of our cloning products, and we also did some controls:

• Colony 10 with no enzyme (well 11)
• Plasmid pET43.1a + enzyme (well 12)
• Plasmid pET43.1a + no enzyme (well 13)

Well 1 to 10: samples from colony 1 to 10
Well 14: ladder

Procedure:

	Volume (μL)
DNA	5
Cut Smart	3
Ndel	1
Xbal	1
H2O	20

• Digestion at 37°C for 15 min
• Addition of 6 µL of loading dye

We deposited all the samples in an agarose gel previously made.
We let the gel run for 10 min at 85 V + 80 min at 125 V.

Results:

Observations: For samples 1 to 11, we can only see DNA streaks. In wells 12 and 13, we can clearly see the plasmid pET43.1a linearized (well 12) and circular (well 13). In the well 14 we can clearly see the ladder.

Interpretations: The streaks observed on the gel are probably due to the genomic DNA contamination. As mentioned the 08.03.2018, there was an error in the Miniprep of pET43.1a + Seq1 + Seq2.

Digestion of pBR322+ Seq5 + Seq6 + Seq7

Aim: Check that the ligation has successfully worked.
Restriction enzyme used: EcoRI

Expected results:

pBR322	linearized	4 kB
pBR322 + Seq5	1 fragment	5.4 kB
pBR322 + Seq5 + Seq6	2 fragments	6.3 kB
pBR322 + Seq5 + Seq6 + Seq7	2 fragments	7 kB

Procedure:

	Volume (μL)
DNA	5
Cut Smart	3
EcoRI	1
H2O	20

• Digestion at 37°C for 15 min
• Addition of 6 µL of loading dye

We digested 6 samples of our cloning products, and we also did some controls.

• Colony 6 with no enzyme (well 7)
• Plasmid pBR322 + enzyme (well 8)
• Plasmid pBR322 + no enzyme (well 9)

Wells 1 to 6: samples from colony 1 to 6
Well 10: ladder

We deposited all the samples in an agarose gel previously made.
We let the gel run for 10 min at 85 V + 90 min at 125 V.
Results:

Observations: The 6 samples are linearized and migrated exactly like the plasmid pBR322 + Enzyme. Moreover, sample 6 without enzyme migrated like pBR322 without enzyme.

Interpretations: This leads to the conclusion that none of the colonies were transformed with the plasmid AND the insert.
/p>

Liquid culture of pET43.1a + Seq1 + Seq2
(See here the liquid culture protocol)

8 colonies from the petri dish (pET43.1a + Seq1,2 from 08.01.2018) were placed in a liquid culture containing:

• 5 mL of LB
• 5 µL of Carbenicillin (25 mg/mL)

Cultures were incubated at 37°C, 180 rpm overnight.

Cloning pBR322 + Seq8
We used the In-Fusion cloning kit provided by Ozyme
in order to clone our insert in the linearized pET 43.1a.

1. We mixed the following volumes into 1.5mL tubes.

Component	Cloning (μL)	Negative control (μL)	Positive control (μL)
Purified Seq8	4	-	2 (control)
Linearized vector	15	1	1 (control)
5X In-Fusion enzyme premix	4	2	2
Deionized water	-	7	5

2. Incubate for 15 minutes in a water bath at 50°C
3. Ligation (See Ligation protocol)
4. Set in ice
5. Transformation of the 3 plasmids into Stellar competent cells (provided with the clonage kit):
   • Competent cell volume: 50 μL
   • DNA volume: 2 μL
6. Culture on petri dishes LB agar plate supplemented with carbenicillin overnight at 37°C