about point mutation

From: Arun Kumar
Date: 24 February 2012 09:05
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

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From: Tim Gruene 

Dear Arun,

it's been a while for my last PCR, so take my comment with caution:
33bases seems very long for a primer especially if it is only for a
point mutation. Could you trim it down to 20-25
bases? I would cut at the downstream side.
Tim

- --
Dr Tim Gruene

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From: Charlotte Hodson

Dear Arun,

I find stratagene method using Pfu turbo enzyme much more successful then using phusion. If you go to their website they even have a tool to design the 'perfect' primer!

Happy cloning!

.

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From: D Bonsor
Phusion requires that the primers are phosphorylated for mutagensis to work, unlike Pfu. If you cannot phosphorylate them use Pfu as recommended by Charlotte

Dan

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From: Dima Klenchin

Phusion requires that the primers are phosphorylated for mutagensis to work, unlike Pfu. If you cannot phosphorylate them use Pfu as recommended by Charlotte

Not really. Phusion *protocol* requires phosphorylated primers but seemingly the only reason for that is that they needed to find a way to bypass Stratagene's patent on "QuikChange" protocol. There is quite obviously no need for ligation at all as evidenced by any QuikChange reaction. (Even more, there is no need for two primers! We *only* ever use a single primer and the frequency of positive clones is the same).

As for the Pfu, there are now much better derivatives of it than the good old Turbo. "Ultra" has lower mutation rate and "Ultra Fusion II" is a lot better in every respect (and in our hands significantly better than Phusion).

- Dima

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From: Xiaodi Yu

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