Tom,
1. reloads on the 3700 - are you using the foil pierce routine? We see very
little formamide/dye breakdown when using foil piercing. We reload after
1-2 days at -20 and get decent data. If a capillary gets overloaded with
too much sample, we bump the formamide up to 50 ul from 10ul in that well
and reload using the same injection parameters with good results.
2. Pop5: I have been in the process of changing to pop5 for over two weeks
now! My first capillary array had over 500 runs on pop6, but I wanted to get
pop5 on a completely fresh capillary array, so decided to change the array
out. Then we couldn't get the array to pressurize, then the cuvette syringe
went out, spatials looked like they had a hole in the middle, high on each
side of the array, low or almost non-existent in the middle. The engineer
replaced the cuvette assembly. We titrated cuvette temperatures across 35-50
degrees, settled on 38 degrees which still showed the usual left to right
fade about 3 to 4 fold. The standards run passed ABI's criteria and I
thought we could at least function on pop5. You can get quite decent data
out of very low signal. We're getting 700 bases regularly from good
customers, sometimes going out to nearly 800. This week however the left to
right fade is much worse and the ABI engineer is chained to the machine here
in Utah till it works. I'm sorely tempted to go back to pop6! In short,
think hard before changing to pop5. (Some machines in our genome center have
tolerated the switch to pop5 very well). The few good runs I have had with
pop5 show a small increase in length of read, perhaps 50 bases, but pop5 has
the potential to really speed up runs therby increasing throughput. If
throughput is not an issue, stick with pop6 for the short term.
Hope this helps, and if anyone has any suggestions for our left to right
signal woes, I'd be glad to hear them.
Margaret Robertson
Director, DNA Sequencing Facility
University of Utah,
4A 432A, SOM
50 North Medical Drive,
Salt Lake City, UT 84132
Tel: 581-4736
Fax: 585-2978
margaret.robertson@hci.utah.edu
http://www.hci.utah.edu/groups/sequencing
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