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 188.8.131.52> 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
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,
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