responses-"to ppt?"

Naomi Thomson Thomson at bms.com
Thu Aug 1 10:29:46 EST 1996


Thank you everyone for all of your responses to my questions concerning 
EtOH precipitation. You've given us numerous leads to follow. I am 
sending a clipping of some of the responses, and later I will report 
what our success rate was. 

OUr next question concerns the upgrade of the sequencers to the 48 and 
64 well format. Ever cautious, I would like to know if anyone is is 
using these upgrades successfully? We would be upgrading a stretch and a 
non-stretch 373.

Here are your responses to the previous question, and Thanks again!

Naomi

Subject: to PPT or not to PPT
To: nobody at net.bio.NET
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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) 252-5967MAIL>   
From:   IN%"hhills at iastate.EDU"
To:     IN%"Thomson at bms.com"
CC:
Subj:   Purification


Subject: Purification
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Naomi,

I get samples for gel runs that have been ethanol precipitated.  They 
are
not as clean as with the CentriSep columns.  We are continuing to use 
the
columns for our facility.  Adjacent lanes are often affected when we run
the ppct samples on the same gel with our other samples.

Hal

Harold G. Hills, Ph.D., DNA Sequencing Specialist       515 294-9585


1184 Molecular Biology Building                         515 294-1597 FAX
Iowa State University                                   
hhills at iastate.edu
Ames, Iowa 50011-3260

    #7          30-JUL-1996 19:36:21.77                                  
NEWMAIL
From:   IN%"ballard at fmppr.fmnh.ORG"  "Bill Ballard"
To:     IN%"nobody at net.bio.NET"
CC:
Subj:   PPT or not to PPT

To: nobody at net.bio.NET
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Hey:

Here is a really simple protocol that we use at The Field Museum in 
Chicago
for precipating 10=B5l cycle sequencing reactions-we do this in 
96--plate
format. We don't get dye "blobs" and we don't loose the small fragments.

1.      Add 40 =B5l ddH2O, 5 =B5l 3 M NaOAc, 125 =B5l Cold EtOH.  Let 
sit 1=
 Hour
at -20=B0C (I am not sure how important this 1 hour at -20=B0C actually 
is-=
-I
have used 30min at -20=B0C without a problem).

2.      Spin for 15 min. Aspirate off the liquid.
3.      Add 200=B5l  70% ethanol.
4.      Spin for 5 min. Aspirate the liquid.
5.      Dry in the speedvac.

Hope this helps:

Cheers

Bill Ballard

From:   IN%"bishop at msvax.mssm.EDU"  "David F. Bishop"
To:     IN%"nobody at net.bio.NET"
CC:
Subj:   RE: to PPT or not to PPT

MAIL>
    #10         31-JUL-1996 06:01:57.18                                  
NEWMAIL
Subject: Re: to PPT or not to PPT
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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




MAIL>
    #1          31-JUL-1996 18:57:21.51                                  
NEWMAIL
                                                  
Our experience with EtOH ppt has been less than pleasing and we  have
reverted back to column purification.  We didn$t have any problems with
T-Blobs but the first 50-60 bp were virtually unusable.  If you can live
with this than go with the EtOH ppt if you$re doing only a few samples.
For a full run we have found that both methods take about the same 
amount
of time and the extra usable data aquired with the column method is 
worth
the extra expence.

Hope this helps :-)


Shaun Tyler
DNA Core Facility
Laboratory Centre for Disease Control
Health Canada

Ph#:  (613) 941-6441
FAX#: (613) 957-1358

E-mail:  styler at hpb.hwc.ca




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