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Team:SDU-Denmark/labnotes
From 2010.igem.org
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''Methods:'' PCR and Gel electrophoresis | ''Methods:'' PCR and Gel electrophoresis | ||
| - | ''Notes:'' Since our FldhDC primers have yet to work, we have decided to test them 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). | + | ''Notes:''Since our FldhDC primers have yet to work, we have decided to test them 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). |
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: | ||
| + | |||
[[Image:Team-SDU-Denmark-PCR_temp_for_FlhDC.png]] | [[Image:Team-SDU-Denmark-PCR_temp_for_FlhDC.png]] | ||
Revision as of 08:12, 12 July 2010
Lab notes (7/12 - 7/18)
Contents |
Group: Flagella
Extraction of psb3k3 plasmids with incerted RFP from E. coli MG1655 (log phase)
Methods: MiniPrep, NanoDrop and gel electrophoresis
Notes: We used 9 ml ON culture, loaded 2ul sample and 8ul agarose loading dye on a 1,5% gel,used a DNA ladder mix (100-10000 nucleotides) as marker
Results: Nanodrop: Tube 1: 30.7 mg/ul Tube 2: 27.4 mg/ul
Gel electrophoresis: No result
--Louch07 15:00, 9 July 2010 (UTC)
Polyferation of FlhDC, FlhD and FlhC genes
Methods: PCR and Gel electrophoresis
Notes:Since our FldhDC primers have yet to work, we have decided to test them 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). 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:
Results: The experiment was succesfull! We could detect FlhDC DNA in the PCR's run between 42.6˚C and 48.3˚C. FlhD DNA in PCR’s run between 40.3˚C and 44.3˚C and also between 48.3˚C and 50.3˚C. FlhC DNA in PCR’s run between 40.3˚C and 50.3˚C.
Group: Photosensor
Group: Retinal
Colony PCR of K274210 in pSB1A2 (transformation from 08/07)
Methods: Colony PCR
Protocol: Modified iGEM colony PCR protocol
Notes: MgCl2 was added as a gradient,sample 1-2 contain 2 ul (0,04255), sample 3-5 2,25ul (0,04787). We mistakenly added 30ul (instead of 17ul) premix to sample 6 - 9, so the concentration of MgCl2 is reduced. Sample 6 contains 2,25ul (0,0375), Sample 7-8 2,5ul (0,04167), Sample 9 2,75ul (0,04583).
Results:
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