to PPT or not to PPT

David F. Bishop bishop at msvax.mssm.edu
Wed Jul 31 03:38:30 EST 1996


Naomi Thomson wrote:

>We have been using the FS enzyme for nearly a year, but have continued to use
>the centrisep columns for purification. Now, in order to cut costs and to
>prepare for future automation of the facility, we are considering ethanol
>precipitation.
>
>There have been numerous communications concerning "T-blobs" and "loss of
>an 80
>base front"  which have us concerned that the switch to EtOH ppt's will
>decrease
>the quality and reliability of the sequencing facility.
>
>I would appreciate hearing the experience, good and bad, others in the
>newsgroup
>have had with this method. What protocols do you use? Has anyone managed to
>automate the dye-terminator reaction from A to Z.
>
>Thanks for your input in advance.
>
>Naomi Thomson
>Thomson at BMS.com
>(609) 252-5967

Dear Naomi,

We have used FS chemistry with CentriSep columns in the past with good
results as you find.  We tried to switch to EtOH precipitation as suggested
by ABI but were not satisfied with the results.  The T-blob was present as
were additional bands at the front from the incompletely removed dye
terminators. We also found the EtOH method to be more labor intensive and
more subject to error - such as losing the pellet - than the desalting
column methods.

We now have worked out a method similar to the one Bruce Roe uses (see his
web site for the full protocol:  http://dna1.chem.uoknor.edu/) but with PE
MicroAmp tubes.  We punch a small hole in the bottom of the tube with a
needle, add a dab of glass beads and then an aliquot of Sephadex G-50
equilibrated in water.  These minicolumns are placed in a PE microAmp
holder and the holder placed over another rack of MicroAmp tubes and
centrifuged in a microtiter plate rotor.  The collection tubes are
SpeedVaced, reconstituted with loading buffer and transfered to the plate
lanes.  We get excellent recovery, no T-blobs and no residual dye
terinators.

As an additional note, we found that prewashing commercial columns with
water usually gave cleaner results than if used without prewash.

Cheers,

David



David F. Bishop, Ph.D.                   | EMail:bishop at msvax.mssm.edu
Dept. of Human Genetics, Box 1203        |
Mount Sinai School of Medicine           | Phone: (212) 241-6946
5th Avenue and 100th Street, NY,NY 10029 | FAX:   (212) 360-1809





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