Re: low sensitivity on ABI476

From: J. Myron Crawford (j.myron.crawford@yale.edu)
Date: Mon Nov 27 2000 - 10:02:09 EST

Hello all,

        Other possibilities for the low sequencing yield are a leaking R3 (100%
TFA) valve and bad ethyl acetate, n-butyl chloride, or n-heptane (S2,
S3, S1). A TFA leak can neutralize the base (TMA or n-methylpiperdine)
so that PiTC cannot couple. We also had a problem with bad ethyl
acetate that fits these symptoms. The ABI organics are distilled across
DTT crystals. In our case, the bad lot of ethyl acetate apparently did
not pick up enough DTT. Low level samples did not sequence at all and
higher level ones did not sequence as well as expected (lower yields and
poor RY). Try adding 20 ul or so of 1 mg/ml DTT to 200 ml of the
organic and see if it helps. If it makes a difference, play with the
amount so you can add as little as possible. The added DTT can cause
artifacts before Asp and Pro.

                                Myron 

-- 
J. Myron Crawford
HHMI Biopolymer Facility
W.M. Keck Biotechnology Resource Laboratory
Yale University

phone: (203) 737-2635
fax:   (203) 737-2638
email:   j.myron.crawford@yale.edu
website: http://info.med.yale.edu/wmkeck

Ken Williams wrote:
> 
> >Date: Thu, 23 Nov 2000 11:09:17 +0100
> >From: Marcus Macht <Marcus.Macht@uni-koeln.de>
> >Subject: Re: low sensitivity on ABI476
> >Sender: Association of Biomolecular Resource Facilities
> > <abrf-request@aecom.yu.edu>
> >X-Sender: akc49@pop.rrz.uni-koeln.de
> >To: Recipients of ABRF List <abrf@aecom.yu.edu>
> >Cc: abrf@aecom.yu.edu
> >X-Mailer: QUALCOMM Windows Eudora Light Version 3.0.6 (32)
> >Old-To: dorian.immler.di@bayer-ag.de
> >
> >At 11:53 22.11.00 +0100, you wrote:
> >>dear all,
> >>
> >>we have some problem with our abi476 which we cannot really identify at the
> >>moment. what we see is an extremely low initial and repetitive yield in our
> >>sequencing (e.g. beta-lacto tests), but the amino acid standard looks
> >fine. all
> >>valves seem to work and the injection and reagent peaks look OK. any ideas
> >what
> >>this might be??????
> >>
> >>thnx in advance for any hints,
> >>
> >>dorian
> >>
> >
> >Hello,
> >
> >we had exactly the same problem some time ago with our 473. The problem was
> >related to the trimethylamin. In our special case the pressurizing valve
> >for R2 was faulty. The valve test showed no error, but the valve did not
> >move (do you hear a "click" from the valve when it switches?). We changed
> >the vent valve (which is not really needed) with the pressurizing valve and
> >the intensity returned to normal values. It is difficult to detect the TMA
> >during the sequencing. The most convenient way is to look for
> >mist-formation in the waste bottle.
> >I hope that this could help you a bit.
> >
> >Yours sincerely,
> >Marcus
> >********************************************************
> >Dr. Marcus Macht
> >University of Cologne
> >Centre for molecular medicine - Service laboratory
> >Joseph-Stelzmann-Str. 52
> >50931 Cologne, Germany
> >Tel.:  +49 221 478-6995
> >Fax:   +49 221 478-6977
> >e-mail: Marcus.Macht@uni-koeln.de
> >**************************************************************************

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