Sheryl- Let's give up on enclosures and I'll try pasting the text of the
protocol directly into the email message.
Protocol for GC rich templates
Full strength BD Mix 8 ul (ABI's BigDye Terminator Ready Reaction Mix)
Template 1 ug
Primer 25 ng=3.2pmol
DMSO 1-2 ul
dH2O qs to 20 ul
Hot start 98C 5min
cycle 40X 98C 40 sec
50C 15sec
60C 4min
hold at 4C.
This worked well for Andy Fire's 80bp GC insert in pUC and for the ABRF
difficult templates. (better than the GTP kit).
Other suggestions for dire circumstances involving GC richness or secondary
structure:
1. dGTP kit reads through Gs well, Cs poorly. Use a primer from either end,
maybe one strand is richer in Gs than the other.
2. Digest out a fragment of interest (the smaller the better), gel purify, and
sequence.
3. PCR out a fragment of interest, gel purify, and sequence.
4. Try different primers. A higher melting temperature primer may help. Go
up to 30bp in length if necessary.
OK, Sheryl, that is the protocol that our core facility distributes to tearful
department members with troublesome templates. I hope it will be helpful to
you. The addition of DMSO seems to be the magic bullet. I'd go with 2ul of
DMSO/rxn. Good luck. Regards, Allison
Allison Pinder
Carnegie Institution of Washington
115 W. University Parkway
Baltimore, MD 21210
(410) 554-1207
pinder@mail1.ciwemb.edu
--------------------------------------
Date: 9/7/99 9:40
To: Allison Pinder
From: Sheryl Christofferson
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Date: Tue, 07 Sep 1999 08:35:14 -0500
From: Sheryl Christofferson <sherylc@omrf.ouhsc.edu>
Organization: Oklahoma Medical Research Foundation
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Subject: Re: DNA Seq
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Allison:
Sorry, your protocol came through as a lot of
nonsense. It must not be translating properly. Can
you send it again?
thanks so much
Sheryl Christofferson
OMRF DNA Sequencing Facility
Allison Pinder wrote:
> Part 1Type: x-binhex4