digesting pcr products

brett brett at BORCIM.WUSTL.EDU
Tue May 20 08:33:10 EST 1997


>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

Didja kill the Pfu between amplification and cutting? Sometimes you really
have to clean PCRs up, say, SDS/ProtK then phenol extract/EtOH ppt. Otherwise
you may be blunting your ends right after cleavage. Another trick frequently
cited is to ligate your undigested PCR into a big linear chain. Then cut and
gel purify. 


Brett Lindenbach
    
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu                             




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