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Team:Exeter/lab book/1gp/wk6
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Revision as of 20:52, 26 September 2012
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Single Gene Plasmids and Enzyme Characterisation: 13th - 17th August 2012 Monday 13th August (9.00) • PCR amplification of WbbC from E.coli BL21(DE3) using 3-step PCR • Adding cultures with ligated WbnK-terminator and WclY-terminator in liquid medium overnight Tuesday 14th August (9.00) • Mini-Prepping of ligated WbnK-terminator and WclY-terminator • Gel Electrophoresis to check fragment sizes of ligated WbnK-terminator, WclY-terminator and PCR product generated from yesterday's PCR attempt Gel Electrophoresis showed that WbnK-terminator mini-prep 1 had worked, WbnK-terminator mini-prep 2 and all WclY-terminator mini-preps plus the PCR product were not discovered. For the PCR product, it was thought that the annealing temperature was too high. Lane 1 = DNA hyperladder, Lane 2 = WbnK-terminator mini-prep 1 (expected: 2 bands at 1003bp and 2070bp), Lane 3 = WbnK-terminator mini-prep 2 (expected: 2 bands at 1003bp and 2070bp), Lane 4 = WclY-terminator mini-prep 1 (expected: 2 bands at 1156bp and 2070bp), Lane 5 = WclY-terminator mini-prep 2 (expected: 2 bands at 1156bp and 2070bp), Lane 6 = WbbC PCR attempt, Lane 7 = DNA hyperladder.
Wednesday 15th August (11.00) • PCR amplification of WbbC from E.coli BL21(DE3) using 3-step PCR • Send off WbnK-terminator for sequencing • Gel Electrophoresis to check fragment sizes of PCR product PCR amplification of WbbC was followed using the same protocol but the annealing temperature was taken down from 65°C to 60°C. Gel Electrophoresis did not show a PCR product and it was thought that the annealing temperature was too high again. Thursday 16th August (13.00) • PCR amplification of WbbC from E.coli BL21(DE3) using 3-step PCR • Adding cultures with ligated WclY-terminator in liquid medium overnight PCR amplification of WbbC was followed using the same protocol but the annealing temperature was taken down from 65°C to 55°C. Gel Electrophoresis did not show a PCR product and it was thought that there was an issue with the BL21 genomic DNA template. Friday 17th August (10.00) • PCR amplification of WbbC and FadR from E.coli BL21(DE3) using 3-step PCR FadR is a transcription factor involved in fatty acid metabolism and was used as a false-positive. Gel Electrophoresis did not show any PCR products and it was thought that there was an issue with the BL21 genomic DNA template. No cultures containing ligated WclY-terminator were found. |
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