Site-directed mutagenesis problem

Sébastien Vigneau via methods%40net.bio.net (by sebastien.vigneau from gmail.com)
Tue Jan 18 07:57:44 EST 2011


Maybe you have a lot of non-specific amplification. Does your template/oligo
sequence contains repeats? Or could your oligos possibly anneal somewhere
else on the template? Increasing the annealing temperature of the PCR
reaction could also help...

Maybe something in the reaction mix (e.g. the enzyme) gets stuck to this
specific sequence. You could check that by doing a phenol/chloroform
purification before to migrate on a gel...

Sebastien


2011/1/18 Pepa Florez Pérez <snipeurope from hotmail.com>

>
> Dear all,
>
> I am writing to you becasue I am experiencing problems to carry out a
> site-directed mutagenesis.
> I use two oligonucleotides 22 nts. long, that hybridize completely but not
> on a single-base located in the middle of the sequence.
>
> What I get after carrying out the PCR is a smear in the gel:
>  - The enzyme is not a problem since the positive control (another plasmid
> same lenght with different oligos did work)
>  - I get a smear no matter which vector I use as template, if I use the new
> oligos, but not the others.
>  - If I increase template concentration I get the same result.
>
> Can you guess what it is going on? What else would you try? I am really
> lost.
> If any of you is interested I can send you a summary picture too.
>
> Thank you very much
> Eva
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