Hi,
I think the 'rubbish in, rubbish out' saying is even more true on the 3700
than 377/373 systems. We can get away with a lot more on our gels than the
3700.
As for the loading/running conditions, we use
run temp: 50C
cuv temp: 50C
sample vol 25 x0.1ul
inj v. 1000
inj time 50sec
In essence only changing the cuvette temp by 10C. This is running POP6 by
the way. It made a bigger difference to our genescan samples. We now get a
very even signal strength across the entire capillary array whereas
previously it fell off a lot on the LHS. I'm happy to say this change has
improved both sequencing and genescan results.
As for the template amounts, this is what we are working on, we routinely
use 0.5 BigDye (v2.0) reactions, even for the cosmids. Even when we
quantitate as accurately as we can sometimes the samples overload sometimes
they do not, the problem we face is if we only use half of the cleaned up
product (instead of diluting after 1 run) we occasionally fall into a low
signal trap...which is much more expensive to repeat the entire reaction
rather than dilute and run again....
Oh, before I go, what is the current state of sequencing with the 3700? Are
users happy? I am getting very good reads at the moment (750+ bases for
pgem standards at less than 1% error), but I have spoken to people seeing
much less than this. Am I likely to see a difference as the array gets much
older, or when I eventually swap arrays can I still expect consistent
results with what I get now (my array is about 100 runs old)? I'm happy to
recommend the product we use to clean up our reactions, but to save any
advertising contact me in person and I'll let you know who the supplier is
(it's a G50 spin plate).
Cheers,
Stuart.
PS What are feelings about Snapshot kits on the 3700?
Stuart Bayliss,
Manager,
Genetics Core Facility,
MRC Clinical Science Centre,
Hammersmith Hospital,
Du Cane Road,
London.
W12 0NN
Tel (lab): 020 8383 3181
Tel (office): 020 8383 8305
Fax: 020 8383 8338
email: stuart.bayliss@csc.mrc.ac.uk
web: http://146.179.68.156
-----Original Message-----
From: Cairo, Al [mailto:Al.Cairo@pfizer.com]
Sent: Thursday, August 24, 2000 10:33 PM
To: Recipients of ABRF List
Subject: RE: Injection on the 3700
Hello Stuart and Andrew,
I agree with Stuart, the problem may lie with Andrew's templates. How does
the control template run? Have you tried looking on agarose gel to get a
feel for the quality and quantity?
Stuart mentioned optimizing front end cleanup and cuvette temp, resulting in
1-2% failure rate. For plasmid sequencing we see about the same using
Sephadex to cleanup our BD CS reactions. I am curious to know the run module
conditions you find optimal. Some of our current conditions using POP6 are:
run temp: 50C
cuv temp: 40C
sample vol 25 x0.1ul
inj v. 1000
inj time 50sec
To avoid overloading (which I assume means too much CS reaction product)why
not tinker with the CS reaction. You might quantitate your templates and
dilute them appropriately prior to CS reaction, or try running a 0.5x or
0.25x CS reaction.
Good sequencing,
Al
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