Trouble cloning synthetic insert

Alex Brands abbrands at artsci.wustl.edu
Fri Mar 8 11:38:31 EST 2002


Hello all,

I'm trying to clone a synthetic insert encoding an HA tag.  I ordered two
oligos that, when annealed, form the coding sequence for the HA tag and
have sticky ends compatible with EcoRI and SacI, like this:


EcoRI sticky end                     SacI sticky end
          AATTXXXXXXXXXXXXXXXXXXXXXXXXXAGCT
              XXXXXXXXXXXXXXXXXXXXXXXXX

I cut the vector with EcoRI and SacI.  The sites are very close to one
another, but the no insert ligation gives ~10
colonies, so I think both enzymes are cutting OK. The ligations with
various concentrations of insert also give ~10 colonies, and none of the
clones are positive.  I've done this before, but using different sites,
and it worked great.  Is one of these restriction sites somehow
incompatible with this strategy?  Any suggestions to get this to work?

Alex




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