Hello Arun:
Actually, I am not sure about " i couldn't get my desire point mutation". You
mean you didn't get the pcr product or you could get the pcr product but there
is no mutation. If you didn't get the PCR,Just lower the annealing temperature
to 55 degree. And try extension temperature 72 or 68 degree. You can get it.
After PCR, using DpnI to treat your PCR product to get rid of the template
plasmid.
Yu Xiaodi
> Date: Fri, 24 Feb 2012 09:05:40 +0000
> From: arungreenlo...@gmail.com
> Subject: [ccp4bb] about point mutation
> To: CCP4BB@JISCMAIL.AC.UK
>
> 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
