Invitro mutagenesis

ChenHA hzhen at freeuk.com
Sat Mar 31 21:00:11 EST 2001



andinesh wrote:
> 
> Hi,
> I have a problem. I have cloned a 1.4kb gene into BamHI site of pSBETa vector,
> I have been trying to do invitro mutagenesis using quickchange mutagenesis kit
> from stratagene, somehow its not working with my plasmid, but it works with
> the control plasmid provided along with the kit. I have tried various
> concentrations of both template as well as primers.
> Could anyone suggest some solutions to this problem, I would be very greatful
> if anyone suggest me some solutions which could solve my problem.

I am not familiar with your plasmid, but one problem that may be 
encountered for Quikchange is that that for larger plasmid it doesn't
work well.  If it is more than say 10 Kb, then I would recommend that
you try some other methods.  If not, then there are a number of things
you could try, for instance, lowering the annealing temperature,
increasing the oligos and vector concentration, extension time etc. 
You can also try other polymerases, although that is something I
haven't tried (you have to be careful with the choice of polymerase). 
If your mutated site has high GC content that may also cause
problems.  





> Thanking you
> 
> Dinesh
> Dept. of Botany
> The University of HongKong
> Hongkong
> andinesh at hkucc.hku.hk
> 
> ---




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