Internal deletions by PCR

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Fri May 12 10:18:56 EST 2000


Hi Ulf,
We do that quite routinely, and the method that works best is what I
call "PCR around the plasmid". The idea is to design two primers that
flank the region to be deleted, but point in "the wrong direction" i.e.
the other way round than those that would amplify the region in PCR.
Perform PCR with a good proofreading enzyme (we use Pfu) and long
extension times.  You´ll get a (blunt ended) fragment of "plasmid minus
deleted region"-size. Gel-isolate the fragment, then ligate.
Important details:
* primers must be 5´phosphorylated and good quality
* use no more than 1ng of template
* it is often useful to destroy the original template after ligation by
digestion with DpnI (which won´t harm the PCR product because it is not methylated)

In most cases, greater than 80% of clones are ok.

Hope this helps,
Frank




"Ulf S. Andersson" wrote:
> 
> Hi,
> I wonder if anybody could tip me on a good and fast way of making
> internal deletions in a plasmid by PCR. I have previously done it by
> introducing new restriction sites by site directed mutagenesis (and then
> digesting and re-ligating) which takes quite a long time... there must
> be a smarter way.
> /Ulf.




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