promega wizard kits and the 373

WAS SHOREWA at UCBEH.SAN.UC.EDU
Wed Oct 5 08:56:29 EST 1994


In article <36t4pl$8bl at cerberus-138.wustl.edu>, HAVILAND at KIDS.WUSTL.EDU
(David L. Haviland, Ph.D.) wrote:

> In <Bibbs-2809941623070001 at 137.131.80.19> Bibbs at scripps.edu writes:
> 
> > I talked with a Promega rep today and she told me that the problem with
> > the Wizard kits and the ABI 373 sequencer is that sometimes the fines from
> > the resin get in the DNA samples and inhibit the reaction.  Her suggestion
> > is to pellet the resin, remove the super and then ethanol ppt.  Has anyone
> > tried this with a sample that has failed?  We will try it tomorrow and
> > I'll post Friday.
> > Lisa Bibbs


I routinely run Wizard mini and midi preps for 373 sequencing using
Dyeterminators.  I elute from the Wizard mini column with 100 ul water, do 
ammonium acetate/ETOH ppt , rinse w/70% etoh and dry completely.  We have a
Savant oligo prep that I use to dry the dna. ( a speed vac would work) 
After an eyeball quantitation on an agarose gel.  I sequence.  Works for 95
% of my sequencing.   I had ran an experiment with 8 qiawell samples and  4
wizard preps.  The qiawell gave a single band of dna where the wizard gave
supercoiled and nicked bands.  The 4 wizards sequenced, the 8 qiawells
didn't.  I only try other prep methods for the 5% of the samples that don't
sequence.  The 5% is usually some weird g-c rich or "we really don't know
what this is" bug, that other labs can't make work either.

Alan Shore

University of Cincinnati DNA Core Facility



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