Team:SDU-Denmark/labnotes
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''Methods:'' [[https://2010.igem.org/Team:SDU-Denmark/protocols#Colony_PCR PCR]] and Gel electrophoresis.<br><br> | ''Methods:'' [[https://2010.igem.org/Team:SDU-Denmark/protocols#Colony_PCR PCR]] and Gel electrophoresis.<br><br> | ||
- | ''Notes:'' | + | ''Notes:'' We decided to test our primers on previously purified cromosomal DNA. Examination of the primers showed that the FlhC reverse primer had a melting temperature of only 45˚C. Therefore we decided to run the samples on a gradient PCR. Simultaneously, we prepared 2 extra samples, isolating FlhD and FlhC, respectively. We did this because we wanted to see if our problems were caused because the combined gene-sequence was to long (932bp).<br> |
Because we just wanted to test our primers in this PCR, we used Taq polymerase, because although it doesn’t proofread, it is remarkably cheaper than Pfu. On the [http://www.fermentas.com/en/products/all/pcr-qpcr-rt-pcr/standard-pcr/ep028-taq-dna-native Fermentas homepage] we found that the annealing temperature for Taq is Tm-5 , which in this case means 40˚C. However, Taq polymerase is not very effective at temperatures under 50˚C so we designed the gradient to lies between 40 and 55˚C. More specifically we chose the following temperatures: | Because we just wanted to test our primers in this PCR, we used Taq polymerase, because although it doesn’t proofread, it is remarkably cheaper than Pfu. On the [http://www.fermentas.com/en/products/all/pcr-qpcr-rt-pcr/standard-pcr/ep028-taq-dna-native Fermentas homepage] we found that the annealing temperature for Taq is Tm-5 , which in this case means 40˚C. However, Taq polymerase is not very effective at temperatures under 50˚C so we designed the gradient to lies between 40 and 55˚C. More specifically we chose the following temperatures: | ||
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--[[User:Louch07|Louch07]] 10:13, 12 July 2010 (UTC) | --[[User:Louch07|Louch07]] 10:13, 12 July 2010 (UTC) | ||
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+ | ===Polyferation og FlhDC with Pfu=== | ||
+ | The purified chromosomal DNA was tested with Pfu; a polymerase with proofreading ability. The PCR was run at 44˚C. We decide to run the PCR at this temperature while it is in the middle of the temperature span we used in the last experiment. | ||
+ | Unfortunately the experiment did not give any results. therefore we have to purified some new chromosomal from the E.coli strain MG1655. | ||
== Group: Photosensor == | == Group: Photosensor == |
Revision as of 08:30, 13 July 2010