Attn: DS DNA Sequencers

Brain Foley brianf at med.uvm.edu
Mon Nov 8 13:44:54 EST 1993


Barbara B Grossmann (dr277 at cleveland.Freenet.Edu) wrote:

: I am interested in any feedback regarding sequencing ds DNA that has been
: purified by the following methods: alk lysis mini prep, Qiagen column, Magip (soory)
: Magic preps.  Is there any problems with faint bands or quantitation
: problems with any of these methods when sequencing. I am from USB and
: will post a summary.Please be as specific as possible when describing
: your results. Have a good day.
: Barb Grossmann
: Mgr Tech Services
: USBR
	
	In my experience any method of ds-DNA purification can give good
results if it is done well.  For instance alkaline lysis miniprep DNA
works fine if the E. coli were healthy, the reagents fresh and a good
yeild of plasmid DNA obtained.  In practice I find miniprep DNA to be
highly variable in quality.  Some of my cultures are usually overgrown
while others are just getting started, so the yield is variable. 
	If I need to sequence several kb from one plasmid, I take the
time to do a CsCl gradient prep after alkaline lysis of a 500 ml
culture.  This is probably overkill, but I know I can count on great
results.  I hate to waste 35-S and the running of a gel only to
find out that some of my reactions did not work well.
	I have developed a feeling that if you have good clean DNA, you 
can get away with other variables being off a bit (ratio of template:primer,
ratio of polymerase:template, temperature of termination reaction, etc.) 
and still get a good gel out of it.  If you have "dirty" DNA it can still
provide good sequence if everything else is just right.
	I have seen what I thought were problems that could only be solved by
using 7-deaza-guanosine (a band in all four lanes a sequence-specific
sites) that apeared reproducibly in reactions from miniprep DNA and did
not apear at all with CsCl purified DNA.  The miniprep DNA looked good
when 7-deaza-guanosine was used, but the CsCl pure DNA looked good even
without using 7-deaza-G.

	It has always been a bit frustrating to me that very small
diferences in technique can sometimes make big diferences in the end
result.  No two people do their alkaline lysis minipreps exactly the
same.  Some add RNase, some don't.  Some do two phenol extractions and a
chloroform extraction, some use phenol:chloroform mix, some don't do any
extraction.  Just the difference between pipetting the aqueous layer to a
new tube, vs removing the phenol from under the aqueous layer can make
a difference.
	The end result is that if one lab tells me "CsCl pure DNA is a
waste of time!  We get great results from crude preps!"  And another
tells me "Crude DNA never works!  We always use Magic preps."  I don't
doubt either one, and don't know what will work best for me in my lab.
Sometimes it comes down to something really stupid, like what brand of
ethanol you use.  Such that in one lab, only preps that do not require
ethanol work well.  That lab may swear that QIAGEN tips are the only 
way to go, when in fact, it has nothing to do with the column matrix.

--
********************************************************************
*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *



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