Weird, bizzare, inexplicable sequencing problem

Sarven Sabunciyan sarven at org.ecc.ubc.ca
Tue Apr 9 22:53:01 EST 1996


Hi!

I gel purified a PCR product and blunt end cloned it into a Stratagene
vector - a derivative of Bluescprit called Direct Script (or something). 
This vector does not have any Eco or Bam sites within it but the primers
I used for the PCR contain these sites (so basically these restriction
sites are present only within the insert).  After cloning, I minipreped a
bunch of clones and cut them with Eco/Bam to ensure that they had inserts
and infact, inserts of the proper size (~170 bps) were present.  Note
that I have also cut these clones with only Eco and with only Bam in
which case the insert does not pop out (as expected.  i.e. both Eco and
Bam sites are present).  Now, the interesting part!!!  When I sequenced
these clones (with a Perkin Elmer Taq cycle sequencing kit) I saw my
vector, followed by my first primer containing the BamHI site (which is
about 40bps), 18 bases of sequence information (i.e. not from either the
primers or the vector so presumably from the PCR product) and then I saw
my vector again. The rest of my sequence and my second primer (the one
with the Eco site) is nowhere to be found.  I mean, I know the EcoRI site
is there - you need it to cut out the insert.  Does anybody out there
have a clue about what's going on? I am absolutely stumped!  

Thanks in Advance

Sarven



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