PCR Experiment

You decide to watch a video to help you visualize how to perform the experiment before you set up your own reaction:

You then proceed to screen the isolated DNA for the noveltin transgene, using PCR. You use a positive control for the reaction, which is DNA isolated from a mouse previously determined to carry the transgene. For your negative control, you use distilled water, instead of a DNA sample. The length of the amplified DNA sample is expected to be 370 base pairs (bp). 

You put together the reaction mixture as follows:

• 1.25μl polymerase buffer A and 1.25 μl polymerase buffer B (KAPA Taq PCR kit)

• 5 μl template DNA

• 1 μl forward and reverse primers, respectively, 

• 0.5 μl of dNTPs (KAPA Taq PCR kit)

• 0.1 μl KAPA Taq DNA polymerase (KAPA Taq PCR kit)

• Final reaction volume of 25 μl with distilled water

You set the PCR machine to the following conditions:

• Denaturation: one cycle at 95oC for 3 min, linked to 35 cycles at 95oC for 1 min

• Annealing: 65oC for 1 min, 72oC for 1 min

• Extension: followed by a final extension at 72oC for 10 min