promega wizard kits and the 373

David L. Haviland, Ph.D. HAVILAND at KIDS.WUSTL.EDU
Tue Oct 4 21:59:01 EST 1994


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

Lisa:  Well... yes and no.  We were having a hit or miss of about 50% when 
it came to sequencing on the 373A using Wizzards.  The only protocol that 
consistently did well for sequencing was the alkaline lysis PEG protocol 
that abounds.  
	What we did (with the aid of Promega Tech services) was to first 1) 
when you vacuum the resin with the 55%EtOh solution, leave it on the vacumm 
for an extra minute.  Extra EtOh, Promega claims, will disrupt cycle 
sequencing.  2)  DO the pre-spin PRIOR to adding your warm TE/H2O - to not 
do so allows extra EtOH and loose resin to come through, and 3) do a final 
EtOH PPT on that 50 ul eluted from the resin after doing the step you were 
told above.  Adding these 3.5 extra steps, we have managed to get upwards 
of 80-90% success in Wizzard sequencing.
	Of course, many now know that Promega has come out with a mini kit 
specific for sequencing on the 373A.

Hope this helps,
David



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