Restriction near DNA ends

DMEIER at mis.mcw.edu DMEIER at mis.mcw.edu
Wed Dec 9 17:23:15 EST 1992


Hello all!
  I'm preparing to do a PCR and I've had some very good suggestions from 
this group in terms of how to sequence the PCR product.
  If I go the route of adding restriction sites at 5' ends of the primers, 
I can directionally clone the PCR fragment into a plasmid.  One 
possibility I considered was to add EcoR I and Hind III ends to to my 
primers, but someone here mentioned that Hind III will not cut a site 
with >10 additional bases.  He cited data on "Cleavage Close to the End 
of DNA Fragments" on p 182 of the New England Biolabs Catalog, 1992.
  Based on this data, I won't add a Hind III site since I don't want to 
add an additional 12 bases on top of it.  Having said that, does anyone 
know if the data in this table is really applicable to the PCR situtation 
since 3' of the site will be hundreds of bases instead of the 8, 10, and 
12 bases fragments tested by Biolabs?
  Can someone please send a small list of restriction enzymes they know 
will cut near a DNA end, and what is the recommended amount of spacer DNA 
to add to the end.
  Thanks very much in advance for your advice.

  Dan Meier
  DMEIER at mis.mcw.edu



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