sequencing problems

Connie Runyan c-runyan at
Fri Mar 21 06:20:03 EST 1997

I am trying to sequence PCR fragments that have been blunt end ligated
into a PCR-Script vector from Stratagene.  I am using a Sequenase T7
sequencing kit for sequencing.  What is happening is that I am getting
bands here and there that show up in two lanes. It is always two lanes
(not across all four) and it only happens in the area of the sequence
where the insert is (the plasmid part of the sequence is fine)  I am
wondering if I might have a heterogeneous mix of insert.  If this is the
case, then this would mean that I had picked two different colonies.  The
kit for cloning that I am using has the problem that all of the colonies
grow very clumped together.  Is this something that I am doing wrong with
the cloning.  Any advice would be very helpfull.  

Thanks, Connie

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