sequensing no problem

Hallgeir Bergum Hallgeir.Bergum at medisin.ntnu.no
Mon Mar 24 11:37:53 EST 1997


Connie Runyan wrote:
> 
> 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


Hi Connie

I think your problem is as you say, that you have cloned a gene family ,
and your clone is contaminated from an other colony. This could be a
problem if you inkubale your cells to long. Then they could send out so
called sattelites. So I think this will not bee a problem if you plate
out a smaler amount (ap. 25-100 ul) and pick the colonies at an early
stage ( may bee 12 houres at 37C).

I have cloned and sequensed a lot og PCR produkts (from gene families)
with use of T-Vektors form In Vitrogen, Novagen and Promega.

Hope you get your sequens!

Vidar



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