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.
"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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