In today's lab we prepared another PCR reaction. The first part of the reaction used the best annealing temperature for the B primers that was determined by doing the last PCR. (~44.0 deg. Celius). We set up this part of the PCR, mixing three 20ul samples, containing 4ul of leaf DNA, and one 20ul sample containing no DNA (negative control). This set of mixtures was set to run at 45.2 degrees celcius. Since this annealing temperature worked well in the initial PCR, we expect to have good results, that will be useable for amplification in a T-Vector.
The second set of samples that we set up were for the A primers. These are being rerun to determine a good annealing temperature. We were unable to see any banding around 800bp on our gel during last lab, suggesting that the annealing temperatures we chose for that run were not sufficient for the A primers. One explaination could be that the lowest annealing temperature was with in 2 degrees celcius of our melting temp. for one of the primers. We prepared three 20ul samples using 4ul of rose petal DNA, and one 20ul sample using no DNA (neg. control). These samples were then set to run at three separate annealing temperatures; 36, 37.7, and 40.8 degrees celcius. We hope to see a DNA band at 800 bp during the next lab when we will perform a gel electrophoresis on these PCR samples.
We also included the positive controls for this PCR, one containing a DNA sample IA7+primers and one containing only primers.
Thursday's lab will be used to run a gel electrophoresis to determine the results/success of this PCR experiment.
Written by Kim Lovik