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Signal Strenghts from spatials and 3700 sensitivity

SMarsh3807 smarsh3807 at aol.com
Wed Feb 14 13:23:40 EST 2001


<< 1. Is there a correlation btween absolute signal strenghts from spatial
dyes and the sensitivity of a 3700 in the context of the array yielding
those signals ?? >>
Yes there is a correlation if you are using a buffer different than the buffer
in the spatial calibration kit. The new buffer to use is 9:1 HiDi Formamide:10x
running buffer. This is the "Signal Variation Assay". Use this buffer at the
various cuvette temps to determine which temperature gives the best signal
across the array. There is a very strong correlation to what you will see when
you run ABI's Long Read standards at the same cuvette temperature. Your Samples
will follow the same pattern.


<< 2. Can varying the cuvette temp sometimes affect the absolute signal
strenghts from  spatial dyes and ( potentially ) therefore the performance
of the 3700; >>
See above, but generally 50C works best, but there are several sites that use
48C or other temps. The recommended procedure is to start the signal variation
assay at 35C then go to 40C, 45C and 50C. Find the temp that works best. You
can then try 1degree changes to find the optimal temp, but it is a lot of work
for generally not much difference.



<< 3. Have you heard of any instances where upgrading to firmware version
1.2.1 dimishes signal strength from spatial dyes and by implication actual
samples? >>
I would go to version 1.2.2. I have not seen or heard of signal problems going
with 1.2.1, but there have been instances of other issues with 1.2.1. 1.2.2
fixes those problems. It will also implement the flat field correction on the
CCD camera if you have that version of CCD.
Hope this helps.
Steve Marsh
Director DNA Sequencing Core Facility @ CalTech
(626)395-2755
(former ABI Service Engineer)



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