Path: utzoo!utgpu!watserv1!watmath!uunet!bionet!PUCC.PRINCETON.EDU!MSALMINEN%FINNPHI
From: MSALMINEN%FINNPHI@PUCC.PRINCETON.EDU (MIKA SALMINEN)
Newsgroups: bionet.molbio.methds-reagnts
Subject: RE: PCR sequencing
Message-ID: <9010020744.AA26173@genbank.bio.net>
Date: 2 Oct 90 08:38:00 GMT
Sender: daemon@genbank.bio.net
Lines: 51


As I think this is of general interest I reply to the net:

Doug,

We at @FINNPHI.BITNET do a lot of PCR-sequencing with reasonable success.
Here's our protocol in which you may find the answears you need:

1. denature 0.6-1.8 pmol of pcr product (purified) by heating 2 min at 100 C in
water.

2. snap cool on ice for 5 min.

3. add 6 pmol primer.

4. add buffer.

5. anneal primer for 10 min at +37 C

6. add enzyme, label and do the labelling for 5 min at +37 C

7. divide to extension/termination reactions and keep at +37 C

8. stop reaction (loading buffer)

There are a few more tricks:

- if your DNA is dirty (as agarose purified) use double amount of enzyme, this
countereffects the agarose inhibitory effect on enzyme activity.

- if you want to read near the primer add manganese buffer from the newest
Sequenase kit (one-tenth of total reaction volume) (Tabor et Richardson:PNAS
1989;86, 4076-4080).

We do our sequencing using the Amersham Multiwell system in a 30 mikrol
reactionvolume, but i know about people using Sequenase with similar success.

Those of you using Klenow outhere....

Forget about ancient inferior systems, T7 rules OK!

DISCLAIMER: I have none whatsoever commercial interests in Ame or USB corp:s,
just forwarding my own personal opinions which do not represent any policies
of this institute.


Hope this helps,

Mika salminen

MSALMINEN@FINNPHI (BITNET)