sequensing no problem
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
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!
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