IUBio

Subcloning Site Directed Mutants

Mike Powell mikepowell at mindspring.com
Wed Jan 31 20:33:01 EST 2001


Jayne,

  Pfu has a very low error rate.  We use Stratagene's mutagenesis system
to make point mutants and it uses Pfu polymerase to copy the entire plasmid
while introducing the mutation.  We have used plasmids up to 10kb with no
problems.  I would drop the subcloning step.

-mike-

<jayni76 at my-deja.com> wrote in message news:958ldi$tj2$1 at nnrp1.deja.com...
> I create a lot of point mutants using site directed mutagenesis.  The
> plasmids are usually around 7kb and I use promega Pfu enzyme.  I have been
> subcloning the mutants after PCR to eliminate other mutations but have
been
> informed that people generally no longer bother as the enzymes are
efficient.
>  If I no longer did the subcloning this would save me a lot of time and
> money!!  Any advice please as I am confused.
>
> Jayne
>
>
> Sent via Deja.com
> http://www.deja.com/







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