Hi, I am trying to PCR 1-2kb target regions from mouse cell genomic DNA.
I have tried using GreenTaq from Qiagen and it works, but when i change to the high fidelity KOD enzyme, the PCR reaction does not work.
My conditions for GreenTaq are:
25ng of gDNA per 25ul reaction
annealing temp 55 degrees , 30s
elongation temp 72 degrees , 1 min 30s
I have basically carried out the same conditions to KOD, but it has not worked so far.
Subsequently, I tried varying the amount of gDNA from 7 to 100ng, increasing KOD enzyme from 1U to 2U, increasing the [Mg2+] from 2.5mM to 3mM, as well as decreasing the annealing temperature to 50 degrees.
Any suggestions on what could be the problem, and what I could do now?