digesting pcr products

Steven Goldberg goldberg at bms.com
Tue May 20 10:03:22 EST 1997


In article <338145DF.5564 at flash.net>, rudnick at flash.net wrote:

> Hello-
> 
> I have been repeatedly trying to clone a pcr product generated from
> primers containg engineered restriction sites.  I am aware that some
> enzymes require more than a few bases downstream to properly cut,
> however I did check a chart for cutting the ends of fragments and these
> enzymes should have sufficient room.  The product was generated with pfu
> and the enzymes are HindIII and HincII.  Any ideas why the product won't
> clone?
> 
> Thank in advance!
> 
> Paul

Despite what the charts may claim, there seem to be certain enzymes that
do not digest the ends of PCR fragments well.  This makes it very
difficult to find the correct ligation product when using the common
expression vectors (in particular).  My soluation in such instances is to
first clone the fragment into a plasmid where there is some type of
selective pressure for inserts...my personal favorite is pZeroI or pZeroII
(Invitrogen).  Then excise the fragment with the original enzymes,
isolate, and clone into the desired vector.

Hope this idea is of some use.

Steve



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