I usually play with the cuvette temperatures by running a "signal
variance assay." I have found that to get the most even signal across
the array from left to right and to get the best intensity, running the
SVA helps find the optimal cuvette temp. In fact, once I have optimized
the signal for each machine, I use the SVA protocol for all my spatial
calibrations. The SVA goes something like this:
Perform spatial calibrations as described in the users manual, except
instead of using the provided loading solution in the kit, use SVA
buffer. SVA buffer is prepared by adding 1 part 10x running buffer to 9
parts HiDi formamide. I usually make 10ml at a time. Run the spatial
with the cuvette temperature at 35, then 40, then 45, and finally 50.
Chose the best looking spatial graph and run with that cuvette
temperature. You can optimize further- say you like 45 and 50, but want
it a little better, try 47. I have found that I am usually satisfied
with 50 degrees for POP6. POP5 I know will behave differently. Once I
have the optimal temp., I use that for the machine for all runs. All of
my machines are running at 50 degrees. Before the cuvette was replaced
on one, it liked 45 the best. It seems to vary a bit from cuvette to
cuvette and from lab to lab, room to room, etc. Once optimized, you
should have the best results with that temp.
If it seems you are still not getting good intensity, I can offer some
other suggestions. I've been through about every problem with these
machines, thought the bent tip thing was new to me.
Harry Bakken
emerGen, Inc.
390 Wakara Way
Salt Lake City, UT 84108
(801) 883-0103 x706
-----Original Message-----
From: Peggy Grow [mailto:grow@genetics.med.harvard.edu]
Sent: Friday, April 20, 2001 1:32 PM
To: Recipients of ABRF List
Subject: 3700 problems
Hello all:
Thanks again for all of your input on my strange problems with loading
on
the 3700. It turns out that Brain Coullahan had the correct answer in
that
our autoloader tips were bent! Thank you, Brian, for suggesting that
possibility and helping to solve our problems. We also replaced the
array,
so it could have conceivably been a combination of the two events
causing
the loading mismatches. I would encourage all 3700 users to check your
autoloader tips frequently by unscrewing them and rolling them to see if
they are bent. Mine weren't bent enough to notice while in the machine,
but
it was very obvious once I took them off and rolled them across the
bench!
By the way, ABI Tech Support, while very helpful, told me that there was
no
way that the tips could be bent or be the cause of the problem!
Have any of you changed the cuvette or run temperatures in the run
module?
What effect did it have on your signal strengths? Do you think that
injection times have an effect on signal strengths? Mine are not as
strong
as I would like and am wondering what I could alter to increase them.
Thanks,
Peggy
Peggy Grow
Biopolymers Facility
Howard Hughes Medical Institute
Harvard Medical School
Warren Alpert Building, rm. 341
200 Longwood Avenue
Boston, Massachusetts 02115
phone: 617/432-7481
This archive was generated by hypermail 2b29 : Tue May 01 2001 - 14:07:05 EDT