G.tigrina Hox gene DthoxC insertion into prokaryote E.coli / (by UNIamCloning)/2011/10/13

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PCR Check of Sample Gt.5-2 Today we ran an electrophoresis gel for the DthoxC amplification of our most highly concentrated sample to date - Gt.5-2. We used a 1.2% agarose concentration in SB buffer. The gel showed only 100bp DNA ladder and positive control. (Fail) In one LAST-ditch effort to salvage this project, we tried ONE MORE PCR setup. This time, we used two different sets of primers - ours, the ones designed to amplify Hox gene DthoxC, and two new sets of primers (called COI-1(F) and COI-2(R)) that have proven to work on virtually any animal. These are primers designed to amplify mitochondrial gene cytochrome oxidase I (COI), and were used by us in this PCR setup as a sort of positive control - we wanted to know whether our amplification failures were a result of botched DNA extractions or poor primer design. DNA samples were run at various annealing temperatures in the ThermoGradient using both sets of primers. The annealing temperatures tried were: 51.0°C, 50.3°C, 47.9°C, 45.4°C, 44.0°C. These temperatures were chosen because 44.0°C was the ideal annealing temperature for COI-1 and COI-2 (as well as in a nice range for 15_DthoxCa_F and 15_DthoxCb_R)and we stopped at 51.0°C to avoid the melting point of the DthoxC primers. --The results of this amplification will be tested via electrophoresis on Tuesday!