Hi all,
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 a capillary
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 associated
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:
http://www.genome.ou.edu/proto.html
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.
Cheers,
--bruce
*************************************************************************
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 instruments at
=> > 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 problems can
=> > 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
=> > [...]
=>
=> Carrie,
=> 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 [1] 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 been getting
=> very good results with v 1.1 [2], but only have a single 3700--so am not a
=> representative sample.
=> Phillip San Miguel
=> Purdue Genomics Core Facility
=>
=>
=> [1] See http://bionet.hgmp.mrc.ac.uk/hypermail/autoseq/autoseq.200001/0003.html
=> [2] See http://bionet.hgmp.mrc.ac.uk/hypermail/autoseq/autoseq.200002/0019.html
=>
=> ---
=>
=>
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