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<h3 style="text-decoration: underline; margin-top: 2em !important;">Calendar Week 06</h3> | <h3 style="text-decoration: underline; margin-top: 2em !important;">Calendar Week 06</h3> | ||
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Oligos/Cells: template2 (template), Noros2 (sense Primer), Noroa2 (antisense Primer) | Oligos/Cells: template2 (template), Noros2 (sense Primer), Noroa2 (antisense Primer) | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/a/aa/T--JMU_Wuerzburg--20180418.pdf">20180418.pdf</a> |
<br> | <br> | ||
Conclusion: No result. | Conclusion: No result. | ||
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<br> | <br> | ||
Oligos/Cells: template2 (template), Noros2 (sense Primer), Noroa2 (antisense Primer) | Oligos/Cells: template2 (template), Noros2 (sense Primer), Noroa2 (antisense Primer) | ||
− | |||
− | |||
<br> | <br> | ||
Conclusion: No result. | Conclusion: No result. | ||
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Oligos/Cells: template2 (template), Noros2 (sense Primer), Noroa2 (antisense Primer) | Oligos/Cells: template2 (template), Noros2 (sense Primer), Noroa2 (antisense Primer) | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/b/b2/T--JMU_Wuerzburg--20180424_1.pdf">20180424_1.pdf</a> |
<br> | <br> | ||
Conclusion: No result. | Conclusion: No result. | ||
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Oligos/Cells: template3, Primer: Noro1s, Noro2.2a | Oligos/Cells: template3, Primer: Noro1s, Noro2.2a | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/f/f1/T--JMU_Wuerzburg--20180507_1.pdf">20180507_1.pdf</a> |
<br> | <br> | ||
Conclusion: Positive results, but the negative water control is also positive. | Conclusion: Positive results, but the negative water control is also positive. | ||
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Oligos/Cells: template3, Primer: Noro1s, Noro2.2a | Oligos/Cells: template3, Primer: Noro1s, Noro2.2a | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/4/40/T--JMU_Wuerzburg--20180507_2.pdf">20180507_2.pdf</a> |
<br> | <br> | ||
Conclusion: Positive results, but the negative water control is also positive. | Conclusion: Positive results, but the negative water control is also positive. | ||
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Oligos/Cells: template3, Primer: Noro1s, Noro2.2a | Oligos/Cells: template3, Primer: Noro1s, Noro2.2a | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/7/74/T--JMU_Wuerzburg--20180507_3.pdf">20180507_3.pdf</a> |
<br> | <br> | ||
Conclusion: Positive results, but the negative water control is also positive. | Conclusion: Positive results, but the negative water control is also positive. | ||
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Oligos/Cells: template3, Primer: Noro1s, Noro2.2a | Oligos/Cells: template3, Primer: Noro1s, Noro2.2a | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/b/b8/T--JMU_Wuerzburg--20180508_1.pdf">20180508_1.pdf</a> |
<br> | <br> | ||
Conclusion: No results. | Conclusion: No results. | ||
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Oligos/Cells: Plasmodiumtemplate, primer: P1s P1a | Oligos/Cells: Plasmodiumtemplate, primer: P1s P1a | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/e/ec/T--JMU_Wuerzburg--20180508_2.pdf">20180508_2.pdf</a> |
<br> | <br> | ||
Conclusion: Positive results, but the negative water control is also positive. | Conclusion: Positive results, but the negative water control is also positive. | ||
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Oligos/Cells: template3, Primer: Noro1s, Noro2.2a, annealing temperature 59° C | Oligos/Cells: template3, Primer: Noro1s, Noro2.2a, annealing temperature 59° C | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/0/0d/T--JMU_Wuerzburg--20180514.pdf">20180514.pdf</a> |
<br> | <br> | ||
Conclusion: Positive results, but the negative water control is also positive. Maybe contaminated primers. | Conclusion: Positive results, but the negative water control is also positive. Maybe contaminated primers. | ||
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Oligos/Cells:<span style="font-style: italic;"> Plasmodium </span>template, primer: P1s P1a | Oligos/Cells:<span style="font-style: italic;"> Plasmodium </span>template, primer: P1s P1a | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/f/f3/T--JMU_Wuerzburg--20180515.pdf">20180515.pdf</a> |
<br> | <br> | ||
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. | 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. | ||
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Oligos/Cells: Plasmodiumtemplate, primer: P1s P1a | Oligos/Cells: Plasmodiumtemplate, primer: P1s P1a | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/8/82/T--JMU_Wuerzburg--20180516.pdf">20180516.pdf</a> |
<br> | <br> | ||
Conclusion: 63 °C temperature too high, 60,6 °C positive result, H2O negative. | Conclusion: 63 °C temperature too high, 60,6 °C positive result, H2O negative. | ||
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Oligos/Cells: template3, Primer: Noro1s, Noro2.2a | Oligos/Cells: template3, Primer: Noro1s, Noro2.2a | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/5/5d/T--JMU_Wuerzburg--20180518.pdf">20180518.pdf</a> |
<br> | <br> | ||
Conclusion: 58 °C best temperature, because of the highest cq difference between template and H2O (5,35). | Conclusion: 58 °C best temperature, because of the highest cq difference between template and H2O (5,35). | ||
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Oligos/Cells: template3, Primer: Noro1sneu, Noro2.2aneu, anneling temperature 58°C | Oligos/Cells: template3, Primer: Noro1sneu, Noro2.2aneu, anneling temperature 58°C | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/e/eb/T--JMU_Wuerzburg--20180601.pdf">20180601.pdf</a> |
<br> | <br> | ||
Conclusion: No results, we have to use the enhancer and activator. | Conclusion: No results, we have to use the enhancer and activator. | ||
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Oligos/Cells: template3, Primer: Noro1sneu, Noro2.2aneu, anneling temperature 58 °C | Oligos/Cells: template3, Primer: Noro1sneu, Noro2.2aneu, anneling temperature 58 °C | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/8/8f/T--JMU_Wuerzburg--20180604.pdf">20180604.pdf</a> |
<br> | <br> | ||
Conclusion: 0,4 µM primer has got the best cq-difference. In the next PCR we will test from 1 mM primer. | Conclusion: 0,4 µM primer has got the best cq-difference. In the next PCR we will test from 1 mM primer. | ||
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Oligos/Cells: template3, Primer: Noro1sneu, Noro2.2aneu, anneling temperature 58 °C | Oligos/Cells: template3, Primer: Noro1sneu, Noro2.2aneu, anneling temperature 58 °C | ||
<br> | <br> | ||
− | Result: : <a href=" | + | Result: : <a href="https://static.igem.org/mediawiki/2018/1/1c/T--JMU_Wuerzburg--20180608.pdf">20180608.pdf</a> |
<br> | <br> | ||
Conclusion: 0,5 µM primer has got the best cq difference. | Conclusion: 0,5 µM primer has got the best cq difference. | ||
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Oligos/Cells: Plasmodiumtemplate, primer: P1sneu P1aneu, annealing temperature 61 °C | Oligos/Cells: Plasmodiumtemplate, primer: P1sneu P1aneu, annealing temperature 61 °C | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/6/61/T--JMU_Wuerzburg--20180611.pdf">20180611.pdf</a> |
<br> | <br> | ||
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<span style="font-style: italic;"> Plasmodium </span>very well. | 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<span style="font-style: italic;"> Plasmodium </span>very well. | ||
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Oligos/Cells: Plasmodiumtemplate, primer: P1sneu P1aneu, annealing temperature 61 °C, 100000, 20000, 4000, 800, 160, 32 particles each approach and DNA of cultured Plasmodium | Oligos/Cells: Plasmodiumtemplate, primer: P1sneu P1aneu, annealing temperature 61 °C, 100000, 20000, 4000, 800, 160, 32 particles each approach and DNA of cultured Plasmodium | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/2/2e/T--JMU_Wuerzburg--20180615.pdf">20180615.pdf</a> |
<br> | <br> | ||
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<span style="font-style: italic;"> Plasmodium </span>very well. There were some pipetting mistakes. | 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<span style="font-style: italic;"> Plasmodium </span>very well. There were some pipetting mistakes. | ||
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Oligos/Cells: templatePf, Primer: Pf1s, Pf1a | Oligos/Cells: templatePf, Primer: Pf1s, Pf1a | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/b/be/T--JMU_Wuerzburg--20180928.pdf">20180928.pdf</a> |
<br> | <br> | ||
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. | 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. | ||
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Oligos/Cells: templatePf, Primer: Pf1s, Pf1a | Oligos/Cells: templatePf, Primer: Pf1s, Pf1a | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/a/a3/T--JMU_Wuerzburg--20180930.pdf">20180930.pdf</a> |
<br> | <br> | ||
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. | 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. | ||
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Oligos/Cells: templateP, primer: P1s, P1a; templatePf, primer Pf1s, Pf1a | Oligos/Cells: templateP, primer: P1s, P1a; templatePf, primer Pf1s, Pf1a | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/a/aa/T--JMU_Wuerzburg--20181001.pdf">20181001.pdf</a> |
<br> | <br> | ||
Conclusion: Kit is ok, positive results for Plasmodium, for<span style="font-style: italic;"> P. falciparum </span>the H2O control is also positive. | Conclusion: Kit is ok, positive results for Plasmodium, for<span style="font-style: italic;"> P. falciparum </span>the H2O control is also positive. | ||
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Oligos/Cells: template (10^4 copies each approach), templateb (20 ng), primer: b1a, b1s | Oligos/Cells: template (10^4 copies each approach), templateb (20 ng), primer: b1a, b1s | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/0/06/T--JMU_Wuerzburg--20181002_gel.pdf">20181002_gel.pdf</a> |
<br> | <br> | ||
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. | 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. | ||
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Oligos/Cells: templateP, primer: P1s, P1a | Oligos/Cells: templateP, primer: P1s, P1a | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/c/cf/T--JMU_Wuerzburg--20181002.pdf">20181002.pdf</a> |
<br> | <br> | ||
Conclusion: Kits from Biozym and highQu have got the same efficiency. The temperature is also good from 57 °C to 63 °C. | Conclusion: Kits from Biozym and highQu have got the same efficiency. The temperature is also good from 57 °C to 63 °C. | ||
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Oligos/Cells: templateb (20 ng), primer: b1a, b1s | Oligos/Cells: templateb (20 ng), primer: b1a, b1s | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/b/b1/T--JMU_Wuerzburg--20181003_gel.pdf">20181003_gel.pdf</a> |
<br> | <br> | ||
Conclusion: band visible | Conclusion: band visible | ||
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Oligos/Cells: templatePf, Primer: Pf1sneu, Pf1aneu, Pf2s, Pf2a, Pf3s, Pf3a | Oligos/Cells: templatePf, Primer: Pf1sneu, Pf1aneu, Pf2s, Pf2a, Pf3s, Pf3a | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/9/91/T--JMU_Wuerzburg--20181005.pdf">20181005.pdf</a> |
<br> | <br> | ||
Conclusion: Negative results for primer 1, positive results for primer 2, High cq for primer 3. | Conclusion: Negative results for primer 1, positive results for primer 2, High cq for primer 3. | ||
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Oligos/Cells: templatePf, Primer: Pf2s, Pf2a | Oligos/Cells: templatePf, Primer: Pf2s, Pf2a | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/6/62/T--JMU_Wuerzburg--20181006.pdf">20181006.pdf</a> |
<br> | <br> | ||
Conclusion: All temperatures have nearly the same cq. | Conclusion: All temperatures have nearly the same cq. | ||
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Protocol: 1,5 % agarose gel | Protocol: 1,5 % agarose gel | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/8/83/T--JMU_Wuerzburg--20181007_gel1.pdf">20181007_gel1.pdf</a> |
<h3>07.10.2018 qPCR probe Plasmodium</h3> | <h3>07.10.2018 qPCR probe Plasmodium</h3> | ||
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Oligos/Cells: templateP, primer: P1s, P1a | Oligos/Cells: templateP, primer: P1s, P1a | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/5/56/T--JMU_Wuerzburg--20181007.pdf">20181007.pdf</a> |
<br> | <br> | ||
Conclusion: We can detect all six samples of the biobrick plasmid. | Conclusion: We can detect all six samples of the biobrick plasmid. | ||
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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 | 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 | ||
<br> | <br> | ||
− | Result: nanodrop: 1: 49,1 ng/µl, 2: 8,4 ng/µl <a href=" | + | Result: nanodrop: 1: 49,1 ng/µl, 2: 8,4 ng/µl <a href="https://static.igem.org/mediawiki/2018/4/42/T--JMU_Wuerzburg--20181007_gel2.pdf">20181007_gel2.pdf</a> |
<h3>07.10.2018 Biobrick repeat 2 of method 1</h3> | <h3>07.10.2018 Biobrick repeat 2 of method 1</h3> | ||
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Oligos/Cells: templateP, primer: P1s, P1a | Oligos/Cells: templateP, primer: P1s, P1a | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/f/f1/T--JMU_Wuerzburg--20181010_1.pdf">20181010_1.pdf</a> |
<br> | <br> | ||
Conclusion: We detect all our five stems of cultured Plasmodia. | Conclusion: We detect all our five stems of cultured Plasmodia. | ||
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Oligos/Cells: templatePf, Primer: Pf2s, Pf2a | Oligos/Cells: templatePf, Primer: Pf2s, Pf2a | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/4/4f/T--JMU_Wuerzburg--20181010_2.pdf">20181010_2.pdf</a> |
− | + | ||
<h3>10.10.2018 qPCR probe Plasmodium</h3> | <h3>10.10.2018 qPCR probe Plasmodium</h3> | ||
Participants: Nicole, Rick | Participants: Nicole, Rick | ||
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Oligos/Cells: templateP, primer: P1s, P1a | Oligos/Cells: templateP, primer: P1s, P1a | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/5/53/T--JMU_Wuerzburg--20181010_3.pdf">20181010_3.pdf</a> |
<br> | <br> | ||
Conclusion: We detect all our five stems of cultured Plasmodia. | Conclusion: We detect all our five stems of cultured Plasmodia. | ||
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Oligos/Cells: templateP, primer: P1s, P1a; templatePf, primer Pf2a, Pf2s | Oligos/Cells: templateP, primer: P1s, P1a; templatePf, primer Pf2a, Pf2s | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/a/a6/T--JMU_Wuerzburg--20181012.pdf">20181012.pdf</a> |
<br> | <br> | ||
Conclusion: Both probes have good results. Multiplexing must be also possible with our two primer/probe pairs. | Conclusion: Both probes have good results. Multiplexing must be also possible with our two primer/probe pairs. | ||
Line 615: | Line 615: | ||
Oligos/Cells: templateP, primer: P1s, P1a; templatePf, primer Pf2a, Pf2s | Oligos/Cells: templateP, primer: P1s, P1a; templatePf, primer Pf2a, Pf2s | ||
<br> | <br> | ||
− | Result: <a href=" | + | Result: <a href="https://static.igem.org/mediawiki/2018/3/3c/T--JMU_Wuerzburg--20181013.pdf">20181013.pdf</a> |
<br> | <br> | ||
Conclusion: Multiplexing is also possible with our two primer/probe pairs. We can detect the cultured<span style="font-style: italic;"> Plasmodia </span>DNA. | Conclusion: Multiplexing is also possible with our two primer/probe pairs. We can detect the cultured<span style="font-style: italic;"> Plasmodia </span>DNA. |
Latest revision as of 11:34, 17 October 2018
Calendar Week 06
07.02.2018 Seeding of HEK293T cells
Participants: Nicole, Rick, Andreas, Maria, CorinnaProtocol: Seed cells
Oligos/Cells: HEK293T
09.02.2018 Transfection of HEK293T cells
Participants: Nicole, Rick, Andreas, Maria, Corinna, BenteProtocol: 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, CorinnaProtocol: Seed cells
Oligos/Cells: HEK293T
13.02.2018 Transfection of HEK293T cells
Participants: Rick, Andreas, Corinna, SusannaProtocol: 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, CorinnaProtocol: 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, FrederikProtocol: Transformation
Calendar Week 09
26.02.2018 Seeding of HEK293T cells
Participants: Nicole, Rick, FrederikProtocol: Seed cells
Oligos/Cells: HEK293T
27.02.2018 Transfection of HEK293T cells
Participants: Nicole, Rick, FrederikProtocol: Transfection with PEI
01.03.2018 Harvest of GFP-RNA
Participants: Nicole, Rick, SusannaProtocol: 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, AnnabelProtocol: Seed cells
Oligos/Cells: HEK293T
18.04.2018 Transfection of HEK293T cells
Participants: NicoleProtocol: Transfection with PEI
18.04.2018 RTqPCR with GFP-RNA
Participants: Nicole, Annabel, RickProtocol: LightCycler® 480 RNA Master Hydrolysis Probes von Roche
18.04.2018 SYBRGreen qPCR Norovirus
Participants: NicoleProtocol: 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, RickProtocol: 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, RickProtocol: Agarosegel
Conclusion: Only marker visible.
20.04.2018 Noro PCR with Taq- and Q-Polymerase (Noro) like qPCR from 18.04.2018
Participants: Nicole, RickProtocol: 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, RickProtocol: 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: NicoleProtocol: 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, RickProtocol: 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: NicoleProtocol: 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, RickProtocol: 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, AnnabelProtocol: 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: NicoleProtocol: 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, RickProtocol: 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, AnnabelProtocol: 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, AnnabelProtocol: 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, RickProtocol: 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, AnnabelProtocol: 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, RickProtocol: 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: NicoleProtocol: 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, RickProtocol: 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: NicoleProtocol: 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, RickProtocol: 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, AnnabelProtocol: 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, AnnabelProtocol: 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, AnnabelProtocol: 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, RickProtocol: 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, RickProtocol: 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, RickProtocol: 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, RickProtocol: 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, RickProtocol: 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, RickProtocol: 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, RickProtocol: 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, RickProtocol: 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, RickProtocol: 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: LuisaProtocol: NEB® PCR Cloning Kit
Result: colonies on the plate
03.10.2018 Biobrick method 2
Participants: Nicole, RickProtocol: 6 Minis inoculate
03.10.2018 Biobrick repeat 2 of method 1
Participants: Nicole, RickProtocol: 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, RickProtocol: 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, RickProtocol: 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, RickProtocol: 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: LuisaProtocol: 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, RickProtocol: 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, RickProtocol: 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, RickProtocol: 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, RickProtocol: 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, RickProtocol: 1,5 % agarose gel
Result: 20181007_gel1.pdf
07.10.2018 qPCR probe Plasmodium
Participants: Nicole, RickProtocol: 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, RickProtocol: 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, RickProtocol: sequencing, inoculate Minis
Calendar Week 41
08.10.2018 Biobrick repeat 2 of method 1
Participants: Nicole, RickProtocol: 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, RickProtocol: ligation with T4 ligase
Result: nanodrop: 1: 49,1 ng/µl, 2: 8,4 ng/µl
10.10.2018 qPCR probe Plasmodium
Participants: Nicole, RickProtocol: 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, RickProtocol: 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, RickProtocol: 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, RickProtocol: 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, RickProtocol: 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.