Direct Seq. of PCR-Products: Problem

Wendy Bedale bedal001 at mc.duke.edu
Fri Apr 7 10:20:48 EST 1995


In article <3m2srp$k2d at rrzs3.uni-regensburg.de>,
c4549 at rbis5.biologie.uni-regensburg.de (Michael Liss        t3173) wrote:

..
> 
> Lately I did a lot of Cycle Sequencing using a modified Perkin Elmer Protocol
>
> 
> I used the same primer that I used for amplification (256x degenerated).
> When I sequence a plasmid with that primer it works just fine.
> But a PCR-Product, purified with QiaQuick columns from an agarose gel,
> doesn't seem to be a good template. On my film i have a stop in every
> lane at every position. But the PCR seemed to be very specific.
> 
> I know that nested primers work better for direct PCR sequencing.
> Unfortunately I have an unpredictable sequence immediately following my
> amplification primers. 
>

I am having a similar problem right now with the same cycle sequencing
kit, but it is not as severe as yours: I can read some of the sequence,
but not too well because of bands in multiple lanes.  I tried gel
purifying my PCR fragment and using that as a template- the gel is running
as I write- and if that doesn't work, I may HPLC purify my template or
simply clone.  My suspicion is that I have small amounts of other stuff
that got amplified in the original PCR, even though it looked pretty clean
on a gel.  Like you, I also don't know enough sequence to do nested PCR- I
am using degenerate primers now as it is!
   Good luck!
         Wendy



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