Direct Seq. of PCR-Products: Problem

Eric C. Anderson anderson at pharmdec.wustl.edu
Mon Apr 10 10:04:39 EST 1995


In article <bedal001-0704951027490001 at byrd.biochem.duke.edu>,
bedal001 at mc.duke.edu (Wendy Bedale) wrote:

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


might i make the following suggestion:  try treating your PCR product with
Exonuclease I and Shrimp Alkaline Phospatase.  there are protocols
outlined in the USB/Amersham catalog and if you are interested i'd be
happy to mail you mine.  the Exo I digests ssDNA to get rid of overhangs,
primers and primer extension products.  the sAP is a pretty standard AP
except that it is completely inactivated after 15'@65oC unlike CIP.  i
have used this protocol with great success in automated cycle sequencing
(ABI 373) which is a notoriously picky protocol.

if i can be of further assistance, let me know.

eric

-- 
"i don't know what caffeine does for you, but i'm pretty sure that without it your head caves in."

eric c. anderson                                 anderson at pharmdec.wustl.edu
dept. of molecular bio. and pharm.               (314)362-3963 (lab)
washington univ. school of medicine              (314)862-2435 (home)
660 s. euclid box 8103                           (314)362-7058 (FAX)
st. louis, mo 63110



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