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
