can any one help me in suggesting that what mistake i have did in my mutagenic
pcr . actually my problem is my primer annealing temperature is 81degree. im
using phusion pol enzyme. i have made many trial, i.e., made annealing at 68
and then followed 2 step pcr method and then added 1micro lit of dmso to
50micro lit of pcr mix etc.. but till now i couldn't get my desire point
mutation. my primer length is about 33 and the mutation id at the centre of the
primer.
can anyone help me what i can improve to get result or what mistake i had
did..
thank you all the members in advance,
cheers,
Arun
