Sequencing from boiling mini-preps?

Bruce Roe broe at aardvark.ucs.uoknor.edu
Sat Aug 6 07:43:00 EST 1994


In article <1994Aug5.160732.8787 at galileo.cc.rochester.edu>, sm010b at uhura.cc.rochester.edu (Jean Valjean) writes...
> 
>Hello,
>	I have a large set of deletion mutants that I need to sequence, and
>I was wondering if it is possible to get sequencing quality DNA off boiling
>mini preps.  Does anyone have any experience with this?  If so, what
>protocol do you use and how good are the results.  Thanks Alot.
> 
> 
>Sean J. McMahon
>University of Rochester
>Department of Biochemistry
>sm010b at uhura.cc.rochester.edu

Sean,
	Presumably you are wishing to sequence off double stranded templates.
If this is the case, and you are using incorporation of label (radioactive
or fluorescence), then you'll either need to be very good in your initial
purification steps (via either alkaline lysis or boiling procedures) OR
follow these early steps with some matrix-based purification.  Personally
we have had very good luck with BioRad's Prep-a-Gene or a home-made version.
However, since you asked the question, buy some of BioRad's stuff and give
their protocol a try.  A similar matrix used to be sold as Magic Mini Prep
but no longer is available from Promega (patent infringement by Promega).
With fluorescent primers on the ABI 373a, we just normally do a alkaline
lysis miniprep without the matrix purification step in the end.
	There has been much discussion of this topic in this news group 
so you might want to search the archives via Gopher to:

Name=Search Bionet and Info-GCG articles (Indiana)
Type=7
Port=70
Path=7/.indices/netnews
Host=fly.bio.indiana.edu

Hope this helps and the usual disclamers regarding conflicts of interest.
Cheers....bruce
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