MSALMINEN%FINNPHI at PUCC.PRINCETON.EDU
Tue Oct 2 03:38:00 EST 1990
As I think this is of general interest I reply to the net:
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
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
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,
MSALMINEN at FINNPHI (BITNET)
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