digesting pcr products

Roderic Fuerst Roderic at biogene.co.uk
Thu May 29 16:49:35 EST 1997


In article <goldberg-2005971103420001 at 140.176.74.203>, Steven Goldberg
<goldberg at bms.com> writes
>> 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?

One possibility is that residual DNA polymerase activity may be damaging
the stick ends generated by digestion. This is one reason why PCR
products can be hard to clone following digestion. Assiduous
purification of PCR product prior to digestion will prevent these
problems arising.

-- 
Roderic Fuerst




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