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
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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
On
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!
On 24/02/2012 10:54, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote:
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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
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
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 +
From: arungreenlo...@gmail.com
Subject: [ccp4bb] about point mutation
To: CCP4BB@JISCMAIL.AC.UK
can any one help me
