Question

what is the purpose of the plasmid control pGAP? What is the of the genomic positive control gDNA? Why is exonuclease I added to the PCR products from the first round of amplification?

Answer 1

1) Purpose of the plasmid control pGAP-

control plasmids (pGAP) always is included in experiment. It help to ensure that the observed result in experiment is due to the independent variable which we are testing. And these results are not  due to some other unintended factor. So these Control plasmids are used to minimize any effects of the non-independent variables.

2) is the of the genomic positive control gDNA?

positive control is one which give always positive results if all the experimental conditions are right. This will test all the reaction contents like master mix, MgCl2 amounts, primer annealing temperature, and extension times.

If positive control does not work, this indicate that something is wrong with your annealing or extension times or temperatures, or something is wrong with your MgCl2 or master mix set up.

If your positive control does work and your test samples do not, - shows all the reactions condition is fine , and thre is problem with your test DNA sample.

3)Why is exonuclease I added to the PCR products from the first round of amplification?

If we want to use products of first PCR reaction or second PCR reaction. Then exonuclease is added to clean up the primers left of the first reaction. This will reduce the overcrowding of molecules in the second PCR.