Sequencing from crude lysates

Alexander Kraev kraev at bc.biol.ethz.ch
Mon Oct 21 14:31:31 EST 1996


In article <326BD1B4.2F6A at amicon.com>, Jack.Leonard at amicon.com wrote:

> A recent article in Biotechniques 21:453-457 (Sept 1996) reports
> the use of heat soaked PCR protocols (HS-PCR) in automated DNA
> sequencing reactions to obviate plasmid DNA template purification, and
> to generate up to 500 bases with insignificant background from crude
> lysates.  Is anyone familiar with this technique and if so, do they
> agree with the authors that the overall quality of DNA sequence is
> considerably improved and that it is becoming unnecessary to improve the
> quality of DNA templates (i.e., use DNA directly from E. coli without
> purification)?  Thank you greatly for your
> response in advance.

I read the article in Biotechniques you mention and should say that it is not
that much different from the one published in Nucleic Acids Res., 19 (23)
by Woodford and Usdin in 1991, which the authors of the present paper do
not mention.
Now it is applied to fluorescent terminator cycle sequencing.  The
non-obvious problem
 with this technique is that the copy number of recombinant plasmids often
changes 
dramatically with certain inserts and in such cases the outcome of
sequencing would
 most likely be very poor;  results may also be better with certain
strains than with the other.
If you are prepared to face these inconsistensies, the method is well
worth a try.
It does not however eliminate the need for plasmid purification kits entirely.

-- 
Alexander Kraev, PhD
Biochemie III, ETHZ Zurich
Phone 41-1-632-31-47
Fax 41-1-632-12-13
e-mail kraev at bc.biol.ethz.ch



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