Dear Bernd,
There is no need to add additional volume to your sequencing reactions
prior to spinning through G-50 (8 of 10 ul will come through consistently
without a detectable loss of signal strength). It will also quicken your
dry down time if you collect into, and speed-vac down in standard
polypropylene v-bottom microplates instead of thermal cycling plates. The
surface area over which you evaporate is much larger with the v-bottom
microplates. I would not overheat the samples when dry, particularly
BigDyes, since this can produce artifacts (hmw dye blobs).
Sincerely yours,
-Jack- (affiliated...see below)
Jack T. Leonard, Ph.D.
Technical Manager, Molecular Biology Group
Millipore Corp.
(978)-762-5294
Bernd Weisshaar <weisshaa@mpiz-koeln.mpg.de> on 03/12/99 09:18:54 AM
To: Recipients of ABRF List <abrf@aecom.yu.edu>
cc: bongartz@mpiz-koeln.mpg.de, buerst@mpiz-koeln.mpg.de,
hirtz@mpiz-koeln.mpg.de, lehmann@mpiz-koeln.mpg.de,
roth@mpiz-koeln.mpg.de, viehoeve@mpiz-koeln.mpg.de (bcc: Jack
Leonard/Bev/NA/Millipore)
Subject: DNA-Seq: 96-well sample prep
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Dear Sequencers,
I would like to know if someone knows a good method to dry down the
sequencing reaction samples after performing the clean-up in Sephadex
G-50/Millipore 96-well columns.
Currently, we have the following protocol:
- 10 microl BD reaction
- add 15 microl ddH2O
- run over 96-column plate (equilibrated in ddH2O)
- elute sample in about 25 microl ddH2O (volume varies a bit)
- dry down samples in the 96-well plate in vacuum
- take up sample in 4 microl sample buffer
- denature and load on gel or cappilar
The problem in our hands with this protocol is the evaporation of the w=
ater
after the column. Can anyone tell me how to speed up the process, eithe=
r by
modifying the whole protocol or by optimising the evaporation step?
Thanks and best regards, Bernd
_____________________________________________________________________
PD Dr. Bernd Weisshaar
MPI fuer Zuechtungsforschung
Carl-von-Linn=E9-Weg 10
50829 Koeln
Germany
Tel. office: +49-221-5062 590 Tel. lab: +49-221-5062 311
Fax: +49-221-5062 313
e-mail: weisshaa@mpiz-koeln.mpg.de
WWW-URL: http://www.mpiz-koeln.mpg.de/~weisshaa/BW-info.html
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