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 +
> From: arungreenlo...@gmail.com
> Subject: [ccp4bb] about point mutation
> To: CCP4BB@JISCM
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
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
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" wrote:
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Dear Ar
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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 02/24
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 o
