DNA sequencing with PCR
skspoidn at uk.ac.rdg.susssys1
Wed Jul 15 07:13:53 EST 1992
I'm trying to set up a "dsDNA cycle sequencing"
Without wishing to anger all the fundamentalists out there,
have you tried a kit? The Promega PCR sequencing kit contains a 35S protocol
(I could mail you a copy if you want), and costs
less than makiing all those fiddly solutions up yourself.
On the subject of kit flames, Can anyone really say that they
know a (reasonably) competent molecular biologist that doesn't
have an understanding of how any kits they use work? I do not
include things like computer assisted sequencing, which is really
a different kettle of fish entirely. I don't think so-called kit
mentality leads to people using kits without understanding the basics.
Heck! What are the basics? Does the average molecular biologist
know why you use 2 vols of ethanol to ppt DNA, but only 1 vol of
isopropanol? or why monovalent cations aid the pptn? Or the chemistry
behind the H-bond interactions in base pairing?
I mean, what is the difference between "Add 0.1 vol 3M NaAc
and 2 vol absolute alcohol"
and "Add 2 uL of solution A and 40 uL of solution B"?
Mike "I'll use any kit, any time" Poidinger
Dept of Microbiology
University of Reading
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