Team:JMU Wuerzburg/Notebook

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Calendar Week 06

07.02.2018 Seeding of HEK293T cells

Participants: Nicole, Rick, Andreas, Maria, Corinna
Protocol: Seed cells
Oligos/Cells: HEK293T

09.02.2018 Transfection of HEK293T cells

Participants: Nicole, Rick, Andreas, Maria, Corinna, Bente
Protocol: Transfection with PEI
Result: a huge number of cells died.
Conclusion: This steps should be repeated.

Calendar Week 07

12.02.2018 Seeding of HEK293T cells

Participants: Rick, Maria, Corinna
Protocol: Seed cells
Oligos/Cells: HEK293T

13.02.2018 Transfection of HEK293T cells

Participants: Rick, Andreas, Corinna, Susanna
Protocol: Transfection with PEI
Result: Two days after transfection half of the cells were alive.
Conclusion: A harvest of GFP-RNA is possible

15.02.2018 Harvest of GFP-RNA

Participants: Rick, Maria, Andreas, Corinna
Protocol: E.Z.N.A. Total RNA Kit I (Omega)
Result: Just a small amount of RNA was isolated
Conclusion: The Experiment should be repeated

Calendar Week 08

22.02.2018 Production of GFP/PUG-RNA

Participants: Rick, Susanna, Frederik
Protocol: Transformation

Calendar Week 09

26.02.2018 Seeding of HEK293T cells

Participants: Nicole, Rick, Frederik
Protocol: Seed cells
Oligos/Cells: HEK293T

27.02.2018 Transfection of HEK293T cells

Participants: Nicole, Rick, Frederik
Protocol: Transfection with PEI

01.03.2018 Harvest of GFP-RNA

Participants: Nicole, Rick, Susanna
Protocol: E.Z.N.A. Total RNA Kit I (Omega)
Result: Just a small amount of RNA was isolated
Conclusion: The experiment should be repeated

Calendar Week 16

17.04.2018 Seed HEK293T cells

Participants: Nicole, Annabel
Protocol: Seed cells
Oligos/Cells: HEK293T

18.04.2018 Transfection of HEK293T cells

Participants: Nicole
Protocol: Transfection with PEI

18.04.2018 RTqPCR with GFP-RNA

Participants: Nicole, Annabel, Rick
Protocol: LightCycler^® 480 RNA Master Hydrolysis Probes von Roche

18.04.2018 SYBRGreen qPCR Norovirus

Participants: Nicole
Protocol: LightCycler^® FastStart DNA Master SYBR Green I Kit (Roche))
Oligos/Cells: template2 (template), Noros2 (sense Primer), Noroa2 (antisense Primer)
Result: 20180418.pdf
Conclusion: No result.

19.04.2018 Repeat SYBRGreen qPCR Norovirus

Participants: Nicole, Rick
Protocol: LightCycler^® FastStart DNA Master SYBR Green I Kit (Roche))
Oligos/Cells: template2 (template), Noros2 (sense Primer), Noroa2 (antisense Primer)
Conclusion: No result.

20.04.2018 Control of qPCR with 1,8% Agarosegel

Participants: Nicole, Rick
Protocol: Agarosegel
Conclusion: Only marker visible.

20.04.2018 Noro PCR with Taq- and Q-Polymerase (Noro) like qPCR from 18.04.2018

Participants: Nicole, Rick
Protocol: PCR with Taq- and Q-Polymerase
Conclusion: We repeated this experiment twice, because we used too much Primer for the first part and did not dilute it 1:10 before use.

20.04.2018 1,8% Agarosegel for PCR with Taq- and Q-Polymerase (Noro)

Participants: Nicole, Rick
Protocol: Agarosegel
Conclusion: Taq and Q-Polymerase give a product. For the Taq control we only see Primer and Q control was not visible.

Calendar Week 17

24.04.2018 Repeat Noro qPCR from 18.04.2018

Participants: Nicole
Protocol: RTqPCR mit Lightcycler EvoScript RNA SYBR Green I Master Kit (Roche)
Oligos/Cells: template2 (template), Noros2 (sense Primer), Noroa2 (antisense Primer)
Result: 20180424_1.pdf
Conclusion: No result.

Calendar Week 18

03.05.2018 Noro RTqPCR

Participants: Nicole, Rick
Protocol: RTqPCR mit Lightcycler EvoScript RNA SYBR Green I Master Kit (Roche)
Oligos/Cells: template3, Primer: Noro1s, Noro2.2a, annealing temperature 54°C
Conclusion: Positive results, but the negative water control is also positive.

04.05.2018 Repeat Noro RTqPCR from03.05.2018

Participants: Nicole
Protocol: RTqPCR mit Lightcycler EvoScript RNA SYBR Green I Master Kit (Roche)
Oligos/Cells: template3, Primer: Noro1s, Noro2.2a, annealing temperature 54°C
Conclusion: Positive results, but the negative water control is also positive.

Calendar Week 19

07.05.2018 Repeat Noro RTqPCR from 03.05.2018 with 57°C annealing temperature

Participants: Nicole, Annabel, Rick
Protocol: RTqPCR mit Lightcycler EvoScript RNA SYBR Green I Master Kit (Roche)
Oligos/Cells: template3, Primer: Noro1s, Noro2.2a
Result: 20180507_1.pdf
Conclusion: Positive results, but the negative water control is also positive.

07.05.2018 Repeat Noro RTqPCR from 03.05.2018 with 58°C annealing temperature

Participants: Nicole, Annabel
Protocol: RTqPCR mit Lightcycler EvoScript RNA SYBR Green I Master Kit (Roche)
Oligos/Cells: template3, Primer: Noro1s, Noro2.2a
Result: 20180507_2.pdf
Conclusion: Positive results, but the negative water control is also positive.

07.05.2018 Repeat Noro RTqPCR from 03.05.2018 with 59°C annealing temperature

Participants: Nicole
Protocol: RTqPCR mit Lightcycler EvoScript RNA SYBR Green I Master Kit (Roche)
Oligos/Cells: template3, Primer: Noro1s, Noro2.2a
Result: 20180507_3.pdf
Conclusion: Positive results, but the negative water control is also positive.

08.05.2018 Repeat Noro RTqPCR from 03.05.2018 with 62°C annealing temperature

Participants: Nicole, Rick
Protocol: RTqPCR mit Lightcycler EvoScript RNA SYBR Green I Master Kit (Roche)
Oligos/Cells: template3, Primer: Noro1s, Noro2.2a
Result: 20180508_1.pdf
Conclusion: No results.

08.05.2018 Plasmodium qPCR from 03.05.2018 with 62°C annealing temperature

Participants: Nicole, Rick, Annabel
Protocol: RTqPCR mit Lightcycler EvoScript RNA SYBR Green I Master Kit (Roche)
Oligos/Cells: Plasmodiumtemplate, primer: P1s P1a
Result: 20180508_2.pdf
Conclusion: Positive results, but the negative water control is also positive.

Calendar Week 20

14.05.2018 Noro RTqPCR with Probe (FAM)

Participants: Nicole, Rick, Annabel
Protocol: Quanti Nova Probe PCR Kit (Quiagen)
Oligos/Cells: template3, Primer: Noro1s, Noro2.2a, annealing temperature 59° C
Result: 20180514.pdf
Conclusion: Positive results, but the negative water control is also positive. Maybe contaminated primers.

15.05.2018 Plasmodium gradient qPCR (annealing temperature 60.6, 63, 65, 66 °C)

Participants: Nicole, Rick
Protocol: RTqPCR mit Lightcycler EvoScript RNA SYBR Green I Master Kit (Roche)
Oligos/Cells: Plasmodium template, primer: P1s P1a
Result: 20180515.pdf
Conclusion: Positive results, the negative water control is also positive, but with a clear difference between the cq value of water and template. Cover of 63 °C and 65 °C were open, so these results are wrong.

16.05.2018 Plasmodium gradient qPCR (annealing temperature 60.6, 63 °C)

Participants: Nicole, Annabel
Protocol: RTqPCR mit Lightcycler EvoScript RNA SYBR Green I Master Kit (Roche)
Oligos/Cells: Plasmodiumtemplate, primer: P1s P1a
Result: 20180516.pdf
Conclusion: 63 °C temperature too high, 60,6 °C positive result, H2O negative.

18.05.2018 Noro probe gradient RTqPCR (annealing 58.2, 59.3, 60.4, 61.3 °C)

Participants: Nicole, Rick
Protocol: Quanti Nova Probe PCR Kit (Quiagen)
Oligos/Cells: template3, Primer: Noro1s, Noro2.2a
Result: 20180518.pdf
Conclusion: 58 °C best temperature, because of the highest cq difference between template and H2O (5,35).

Calendar Week 22

01.06.2018 Noro cDNA Synthese, PCR, Agarosegel

Participants: Nicole
Protocol: cDNA Synthese with M-MulV RT Quick Protocol from NEB, PCR with Taq, 1,8 % Agarosegel
Conclusion: no result.

01.06.2018 Noro Probe RTqPCR

Participants: Nicole, Rick
Protocol: LightCycler^® 480 RNA Master Hydrolysis Probes (Roche), primer concentration 0,4 µM, 0,2 µM, 0,1 µM, template 10^4 copies per PCR reaction
Oligos/Cells: template3, Primer: Noro1sneu, Noro2.2aneu, anneling temperature 58°C
Result: 20180601.pdf
Conclusion: No results, we have to use the enhancer and activator.

Calendar Week 23

04.06.2018 Noro RTqPCR

Participants: Nicole
Protocol: LightCycler^® 480 RNA Master Hydrolysis Probes (Roche), primer concentration 0,4 µM, 0,2 µM, 0,1 µM, template 10^4 copies per PCR reaction
Oligos/Cells: template3, Primer: Noro1sneu, Noro2.2aneu, anneling temperature 58 °C
Result: 20180604.pdf
Conclusion: 0,4 µM primer has got the best cq-difference. In the next PCR we will test from 1 mM primer.

04.06.2018 Noro RTqPCR

Participants: Nicole, Annabel, Rick
Protocol: LightCycler^® 480 RNA Master Hydrolysis Probes (Roche), primer concentration 1 µM, 0,5 µM, 0,25 µM, 0,125 µM, template 10^4 copies per PCR reaction
Oligos/Cells: template3, Primer: Noro1sneu, Noro2.2aneu, anneling temperature 58 °C

08.06.2018 Repeat Noro RTqPCR

Participants: Nicole, Rick, Annabel
Protocol: LightCycler^® 480 RNA Master Hydrolysis Probes (Roche), primer concentration 1 µM, 0,8 µM, 0,6 µM, 0,4 µM template 10^4 copies per PCR reaction
Oligos/Cells: template3, Primer: Noro1sneu, Noro2.2aneu, anneling temperature 58 °C
Result: : 20180608.pdf
Conclusion: 0,5 µM primer has got the best cq difference.

Calendar Week 24

11.06.2018 Plasmodium qPCR with genomic DNA cultured Plasmodium

Participants: Nicole, Rick, Annabel
Protocol: RTqPCR mit Lightcycler EvoScript RNA SYBR Green I Master Kit (Roche)
Oligos/Cells: Plasmodiumtemplate, primer: P1sneu P1aneu, annealing temperature 61 °C
Result: 20180611.pdf
Conclusion: Positive results, the negative water control is also positive, but with a clear difference between the cq value. We can detect the genomic DNA of cultured Plasmodium very well.

15.06.2018 Plasmodium standard curve

Participants: Nicole, Rick, Annabel
Protocol: RTqPCR mit Lightcycler EvoScript RNA SYBR Green I Master Kit (Roche)
Oligos/Cells: Plasmodiumtemplate, primer: P1sneu P1aneu, annealing temperature 61 °C, 100000, 20000, 4000, 800, 160, 32 particles each approach and DNA of cultured Plasmodium
Result: 20180615.pdf
Conclusion: Positive results, the negative water control is also positive, but with a clear difference between the cq value. We can detect the genomic DNA of cultured Plasmodium very well. There were some pipetting mistakes.

Calendar Week 39

28.09.2018 gradient qPCR Plasmodium falciparum

Participants: Nicole, Rick
Protocol: LightCycler^® EvoScript RNA SYBR® Green I Master (Roche), primer concentration 0,4 µM template 10^5copies per PCR reaction, annealing temperature 58,5°C, 61°C, 63,7°C
Oligos/Cells: templatePf, Primer: Pf1s, Pf1a
Result: 20180928.pdf
Conclusion: Positive results, but H2O control is near the template cq for 58,5°C. For the higher temperature we did not got a result.

28.09.2018 Biobrick method 1

Participants: Nicole, Rick
Protocol: PCR with taq and Q5 polymerase, annealing temperature 59°C; double approach, one approach digested, the other approach purified with the innuPREP Gel Extraction Kit (Analytic Jena); digest with EcoRI-HF and PSTI-HF
Oligos/Cells: templateP, primer: b1a, b1s

30.09.2018 Biobrick method 1

Participants: Nicole, Rick
Protocol: purified with the innuPREP Gel Extraction Kit (Analytic Jena); measure DNA concentration at nano drop; ligation with T4 ligase
Oligos/Cells: templateb, primer: b1a, b1s
Result: nanodrop: vector: 19,8 ng/µl, taq-1: 8,9 ng/µl, taq-2: 9,9 ng/µl, Q5-1: 14,3 ng/µl, Q5-2: 7,2 ng/µl

30.09.2018 gradient qPCR Plasmodium falciparum

Participants: Nicole, Rick
Protocol: LightCycler^® EvoScript RNA SYBR® Green I Master (Roche), primer concentration 0,4 µM template 10^5copies per PCR reaction, annealing temperature 57,3°C, 59,3°C, 61°C
Oligos/Cells: templatePf, Primer: Pf1s, Pf1a
Result: 20180930.pdf
Conclusion: Positive results, but H2O control is near the template cq for 57,3°C. For the higher temperature we did not got a result.

Calendar Week 40

01.10.2018 Biobrick method 1

Participants: Nicole, Rick
Protocol: transformation
Oligos/Cells: E. coli DH5α
Result: no colonies
Conclusion: The bacteria were not competent, but maybe the digest or ligation were also false.

01.10.2018 qPCR probe (FAM) Plasmodium

Participants: Nicole, Rick
Protocol: LightCycler^® 480 RNA Master Hydrolysis Probes (Roche), primer concentration 0,4 µM, probe concentration 0,2 µM, template 10^6, 10^2 copies per PCR reaction, annealing temperature 61 °C
Oligos/Cells: templateP, primer: P1s, P1a

01.10.2018 gradient qPCR SyBr Green P. falciparum and Plasmodium

Participants: Nicole, Rick
Protocol: LightCycler^® EvoScript RNA SYBR® Green I Master (Roche), primer concentration 0,4 µM, , template 10^5 copies per PCR reaction, annealing temperature 56 °C, 56,7 °C, 58 °C, 61 °C
Oligos/Cells: templateP, primer: P1s, P1a; templatePf, primer Pf1s, Pf1a
Result: 20181001.pdf
Conclusion: Kit is ok, positive results for Plasmodium, for P. falciparum the H2O control is also positive.

02.10.2018 Biobrick repeat 1 of method 1

Participants: Nicole, Rick
Protocol: PCR with Taq polymerase, annealing temperature 77 °C; purified with the innuPREP Gel Extraction Kit (Analytic Jena), before take 5µl for a control on a agarose gel; 2 % agarose gel
Oligos/Cells: template (10^4 copies each approach), templateb (20 ng), primer: b1a, b1s
Result: 20181002_gel.pdf
Conclusion: annealing temperature too high. No band for the templateP, and a little band for template. It must be only the template amount from the beginning.

02.10.2018 gradient qPCR probe (FAM) Plasmodium

Participants: Nicole, Rick
Protocol: One Step RT qPCR Probe ROX L Kit (highQu), Biozym Probe qPCR Kit, primer concentration 0,4 µM, probe concentration 0,2 µM, annealing temperature 57 °C, 58,6 °C, 61 °C, 63 °C
Oligos/Cells: templateP, primer: P1s, P1a
Result: 20181002.pdf
Conclusion: Kits from Biozym and highQu have got the same efficiency. The temperature is also good from 57 °C to 63 °C.

02.10.2018 Biobrick method 2

Participants: Luisa
Protocol: NEB® PCR Cloning Kit
Result: colonies on the plate

03.10.2018 Biobrick method 2

Participants: Nicole, Rick
Protocol: 6 Minis inoculate

03.10.2018 Biobrick repeat 2 of method 1

Participants: Nicole, Rick
Protocol: PCR with taq polymerase, annealing temperature 64°C; purified with the innuPREP Gel Extraction Kit (Analytic Jena), before take 5µl for a control on a agarose gel; 2% agarose gel; digest with EcoRI-HF and PSTI-HF
Oligos/Cells: templateb (20 ng), primer: b1a, b1s
Result: 20181003_gel.pdf
Conclusion: band visible

04.10.2018 Biobrick method 2

Participants: Nicole, Rick
Protocol: 6 Minis inoculate
Result: Minis did not grow. Again 6 Minis inoculate. 05.10.2018 Also did not grow. Repeat NEB® PCR Cloning Kit

04.10.2018 Biobrick repeat 2 of method 1

Participants: Nicole, Rick
Protocol: purified with the innuPREP Gel Extraction Kit (Analytic Jena); measure with the nanodrop; ligation with T4 ligase, transformation
Oligos/Cells: E. coli DH5α, two different approaches
Result: nanodrop: 1: 19,2 ng/µl, 2: 15,4 ng/µl

04.10.2018 Biobrick repeat 2 of method 1

Participants: Nicole, Rick
Protocol: 6 Minis inoculate; 3 idis from positive control BBa_J04450 in pSB1C3 inoculte
Oligos/Cells: E. coli DH5α, two different approaches

05.10.2018 Repeat Biobrick method 2

Participants: Luisa
Protocol: NEB® PCR Cloning Kit
Result: 03.10.2018 colonies on the plate

05.10.2018 qPCR SyBr Green P. falciparum with tree different primers

Participants: Nicole, Rick
Protocol: LightCycler^® EvoScript RNA SYBR® Green I Master (Roche), primer concentration 0,4 µM template 10^5copies per PCR reaction, annealing temperature 58 °C
Oligos/Cells: templatePf, Primer: Pf1sneu, Pf1aneu, Pf2s, Pf2a, Pf3s, Pf3a
Result: 20181005.pdf
Conclusion: Negative results for primer 1, positive results for primer 2, High cq for primer 3.

06.10.2018 Biobrick repeat 2 of method 1

Participants: Nicole, Rick
Protocol: innuPREP Plasmid Mini Kit; nanodrop; test digest, colony PCR, 1,7 % agarose gel
Result: nanodrop: 1: 44,9 ng/ µl, 2: 47 ng/µl, 3: 51,7 ng/µl, 4: 33 ng/µl, 5: 28,2 ng/µl, 6: 55,4 ng/µl 20181006_gel.pdf

06.10.2018 Repeat Biobrick method 2

Participants: Nicole, Rick
Protocol: GenElute Plasmid DNA Midiprep Kit for the vector, nanodrop, digest with PSTI-HF, inoculate 7 Minis from insert

06.10.2018 gradient qPCR SyBr Green P. falciparum

Participants: Nicole, Rick
Protocol: LightCycler^® 480 SYBR Green I Master (Roche), primer concentration 0,4 µM template 10^5, 10^2 copies per PCR reaction, annealing temperature 57 °C, 59,1 °C, 61 °C
Oligos/Cells: templatePf, Primer: Pf2s, Pf2a
Result: 20181006.pdf
Conclusion: All temperatures have nearly the same cq.

07.10.2018 Repeat Biobrick method 2

Participants: Nicole, Rick
Protocol: 1,5 % agarose gel
Result: 20181007_gel1.pdf

07.10.2018 qPCR probe Plasmodium

Participants: Nicole, Rick
Protocol: Biozym Probe qPCR Kit, primer concentration 0,4 µM, probe concentration 0,2 µM, annealing temperature 61 °C
Oligos/Cells: templateP, primer: P1s, P1a
Result: 20181007.pdf
Conclusion: We can detect all six samples of the biobrick plasmid.

07.10.2018 Repeat Biobrick method 2

Participants: Nicole, Rick
Protocol: innuPREP Plasmid Mini Kit of the insert, nanodrop, digest insert, digest vector with EcoRI-HF later PSTI-HF and PSTI-HF and later EcoRI-HF; 1,5 % agarose gel
Result: nanodrop: 1: 49,1 ng/µl, 2: 8,4 ng/µl 20181007_gel2.pdf

07.10.2018 Biobrick repeat 2 of method 1

Participants: Nicole, Rick
Protocol: sequencing, inoculate Minis

Calendar Week 41

08.10.2018 Biobrick repeat 2 of method 1

Participants: Nicole, Rick
Protocol: innuPREP Plasmid Mini Kit, nanodrop
Result: nanodrop: 1: 76,9 ng/µl, 2: 53,0 ng/µl, 3: 61,2 ng/µl, 4: 51,2 ng/µl, 5: 54,1 ng/µl, 6: 40,2 ng/µl

08.10.2018 Repeat Biobrick method 2

Participants: Nicole, Rick
Protocol: ligation with T4 ligase
Result: nanodrop: 1: 49,1 ng/µl, 2: 8,4 ng/µl

10.10.2018 qPCR probe Plasmodium

Participants: Nicole, Rick
Protocol: Biozym Probe qPCR Kit, primer concentration 0,4 µM, probe concentration 0,2 µM, annealing temperature 61 °C, template 10^5, 1*10^4, 4000, 800, 160, 32, 6,4 copies per PCR reaction
Oligos/Cells: templateP, primer: P1s, P1a
Result: 20181010_1.pdf
Conclusion: We detect all our five stems of cultured Plasmodia.

10.10.2018 qPCR SyBr Green P. falciparum

Participants: Nicole, Rick
Protocol: LightCycler^® 480 SYBR Green I Master (Roche), primer concentration 0,4 µM template 10^5, 1*10^4, 4000, 800, 160, 32, 6,4 copies per PCR reaction, annealing temperature 61°C
Oligos/Cells: templatePf, Primer: Pf2s, Pf2a
Result: 20181010_2.pdf

10.10.2018 qPCR probe Plasmodium

Participants: Nicole, Rick
Protocol: Biozym Probe qPCR Kit, primer concentration 0,4 µM, probe concentration 0,2 µM, annealing temperature 61°C, template 10^5, 1*10^4, 4000, 800, 160, 32, 6,4 copies per PCR reaction
Oligos/Cells: templateP, primer: P1s, P1a
Result: 20181010_3.pdf
Conclusion: We detect all our five stems of cultured Plasmodia.

12.10.2018 qPCR probe (FAM) Plasmodium and probe (HEX) P. falciparum

Participants: Nicole, Rick
Protocol: Biozym Probe qPCR Kit, primer concentration 0,4 µM, probe concentration 0,2 µM, annealing temperature 61°C, template 10^5, 1*10^4, 4000, 800, 160, 32, 6,4 copies per PCR reaction
Oligos/Cells: templateP, primer: P1s, P1a; templatePf, primer Pf2a, Pf2s
Result: 20181012.pdf
Conclusion: Both probes have good results. Multiplexing must be also possible with our two primer/probe pairs.

13.10.2018 Multiplex qPCR probe (FAM) Plasmodium and probe (HEX) P. falciparum

Participants: Nicole, Rick
Protocol: Biozym Probe qPCR Kit, primer concentration 0,4 µM, probe concentration 0,2 µM, annealing temperature 61°C, template 10^5, 1*10^4, 4000, 800, 160, 32, 6,4 copies per PCR reaction
Oligos/Cells: templateP, primer: P1s, P1a; templatePf, primer Pf2a, Pf2s
Result: 20181013.pdf
Conclusion: Multiplexing is also possible with our two primer/probe pairs. We can detect the cultured Plasmodia DNA.