Hi,
It is not necessary to take a hit on read length when you go to 96 lanes.
We run 36 cm 2x runs for our regular clients (off the street samples I call
them). Our pGEM controls run 680 - 740 bases machine called 98.5% accurate.
For our EST project we run 36 cm 4x and the pGEMs have a couple of errors in
the first 20 bases but then 100% to the end of the run (480 - 510 bases).
The same for the EST samples even after a 30 - 40 base run of T's the good
samples run to the end without an N.
We are using the membrane combs from the Gel Company. They are great because
you can load at the bench rather than standing at the machine. We use the
regular glass plates not the step plates. We have fewer problems with tracking
than we did with the 64 lane sharks tooth. If done right the membrane combs
give very sharp separation between the lanes.
We tried the ficoll method but did not like the resolution in the later part
of the run. Early on using water the lanes ran together in the early part
of the run due to diffusion of the smaller fragments. To avoid this you need
to set up and start a run so that all you have to do is open the door, slide
the comb in and immediately close the door to run the samples in for 45 sec.
If you bend a tooth on the comb forget it. Trying to take the comb out and
put it back takes too long and you will get diffusion. After running the
samples into the gel remove the comb and add buffer. To bring the machine
up to temperature we start a prerun and then pause so we get heating and
circulation but no current.
If you would like to see gel images of some of our EST runs go to:
http://biotech.missouri.edu/dnacore/gelimage
Joe Forrester
DNA Core Facility
University of Missouri
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