DNA sequencing with PCR

Mike Poidinger skspoidn at uk.ac.rdg.susssys1
Wed Jul 15 07:13:53 EST 1992


re:
I'm trying to set up a "dsDNA cycle sequencing"
 protocol...

Paulo

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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