DNAseq: POP5 on 3700
weisshaa at mpiz-koeln.mpg.de
Tue Mar 7 02:50:45 EST 2000
one additional question for the 3700 specialists:
When switching to v1.1 and POP5, were does base calling start (in
bases away from the primer)? On the runs I have seen, the first about
70 or so bases are lost due to mobility shifts. Our single 3700 is
now running with v1.1, but we still use POP6. Has the problem been
solved or is it anyhow only roumors?
> I've been noticibly quiet on this topic because we only last
>week began the conversion to pop5. We now have converted one of
>our 3700's to pop5 and done several experiments playing with the capillary
>and cuvette chamber temperature. With pop6 we have been running at
>chamber temperature of 40 deg C and cuvette sheath flow temperature of 35.
>The defaults for these two is higher than this so we ran the same samples
>at these setting and at the default settings. The results convinced us that
>the lower temperatures were better than the default as we got ~50 phred20
>bases more at 40/35 deg C than at the default settings.
> Since we also had increased the voltage by 250 volts with pop6, we
>now are going to compare the same samples at the default of 6500 volts and
>at an increased setting of 6750 volts. Should have these results by Monday.
>These are the only settings we are contemplating tampering with for pop5 at
>this time, but the experience so far has convinced us to begin switching the
>other 4 3700s to pop5 this week. Fortuantely, 'knock on wood', all 5 have
>been up and running all week except for one that ran and didn't seem to see
>or collect any signal (eventhough re-running the same plate gave beautiful
>signal on the same instrument after restarting both the 3700 and the
>computer). We routinely re-start both the 3700 and computer at least once
>a day just to free the air of any funkey electrons that might have crept in.
>Eventhough ABI says not to do this, I feel better knowing that the memory
>is purged by restarting.
> On a related note, last week I updated and posted our latest template
>isolation protocols and they can be found at URL:
>The only other things to comment on is that we're dissolving our SephadexG-50
>purified, dried down, wrapped in Al foil and stored frozen reactions only in
>dd-water, using, as I posted earlier, increased amounts of water in those
>plates which have to sit longer in the instrument before loading. We also put
>some Al foil over the plexaglass portion of the door to cut down on any stray
>light that may enter the sample chamber just because I'm slightly paranoid
>about light effects on the dyes.
>Lastly, we're doing all our sequencing reactions in 5-7ul with 1/12th the
>amount of BigDye mix, see URL: http://www.genome.ou.edu/big_dyes_plasmid.html
>using 60 cycles.
>Bruce A. Roe, Ph.D George Lynn Cross Research Professor
> Department of Chemistry and Biochemistry
> University of Oklahoma, Norman, OK 73019-0370, U.S.A.
>Phone: (405) 325-4912 or 7610; FAX: (405) 325-7762; e-mail: broe at ou.edu
>********************** http://www.genome.ou.edu/ ************************
> You write:
>=> Date: Fri, 03 Mar 2000 18:48:30 +0000
>=> From: Phillip San Miguel <pmiguel at purdue.edu>
>=> Subject: Re: Loss of resolution on 3700, and failed lanes
>=> Sender: owner-autoseq at hgmp.mrc.ac.uk
>=> To: autoseq at net.bio.net
>=> Carrie Sougnez wrote:
>=> > Hello Grace,
>=> > I am from the MIT/Whitehead Institute Genome Center. We
>also run 8 plates
>=> > a day (really 2 - 384well plates/day) on the 3700. We run 123
>=> > a time. We too see a wide range of high and low quality data
>on the 3700.
>=> > We can usually attribute low quality data to a internal problem in the
>=> > instrument. For example plumbing, camera, and laser alignment
>=> > all cause low quality data. What types of maintenance
>protocols are you using
>=> > to keep your instruments performing?
>=> > We also see discrepancy in data quality between the left and
>right side of the
>=> > array (a difference of 100 phred 20 bases from left to right). This
>=> > correlates to the signal intensity from left to right, but
>there is no fix for
>=> > this yet... we have had limited success in changing the
>temperature of the
>=> > cuvette during run modules.
>=> > As for your resusupension solution, you may want to try 0.5mM
>EDTA. We tested
>=> > water, formamide, 12.5% pyrillidinone, and EDTA. The 0.5mM
>EDTA gives us the
>=> > longest read lengths.
>=> > Carrie Sougnez
>=> > Coordinator, Sequence Detection
>=> > Whitehead Institute Center for Genome Research
>=> > [...]
>=> Thanks for posting your comments here.
>=> By the way, what % of your machines have you switched to
>version 1.1 data
>=> collection software? I ask because Kevin McKernan posted Jan. 9th
> saying at
>=> Whitehead they were limiting the number of machines converted to
>v. 1.1 because of
>=> various problems. I was wondering if those problems had been
>cleared up or not.
>=> Also it might explain your increase in read length with 0.5 mM
>EDTA if you were
>=> not using foil piercing (which was implemented in v. 1.1). I have
>=> very good results with v 1.1 , but only have a single 3700--so am not a
>=> representative sample.
>=> Phillip San Miguel
>=> Purdue Genomics Core Facility
>=>  See
>=>  See
PD Dr. Bernd Weisshaar
MPI fuer Zuechtungsforschung
Tel. office: +49-221-5062 590 Tel. lab: +49-221-5062 311
Fax: +49-221-5062 313
mailto:weisshaa at mpiz-koeln.mpg.de
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