Andrew,
What sort of failures are you seeing? I've not had to change very much on
the module really, apart from optimising the cuvette temperature (which made
a HUGE difference)...is it overloading of samples, or complete failures (no
injection)? Could be too many ions in the loading water/formamide/whatever.
Any chance of seeing a chromatogram image?
I spent a lot of time & effort optimising the front end of the pipeline (ie
get a really good reaction clean up before you get anywhere near the 3700).
This made the greatest difference in what we see and dropped our failure
rate to 1-2%. I'm quite confident now that any failures usually result from
bad DNA/primer preps we are sent, which tends to be the case, rather than
the injection itself. Of course this is assuming the injection procedure is
working OK (EPT fine, no blocked needles etc)
As an aside do you (or any 3700 users) see a regular overloading once
plasmids get to the 6-7kb range? (a re run after of 5ul of remaining loading
solution made up to 10ul usually give great results). We see it all the
time, yet our cosmid sequencing (40kb) runs like a dream with very few
overloads. I'm looking into it now but if anyone has input I'd be glad to
hear it....
Regards,
Stuart
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: Andrew.Walding@astrazeneca.com
[mailto:Andrew.Walding@astrazeneca.com]
Sent: Thursday, August 24, 2000 10:59 AM
To: Recipients of ABRF List
Subject: Injection on the 3700
Has anyone done much optimising of injection voltage and times on the 3700?
What are your conclusions? We would like to optimise ours for plasmid
sequencing, as we are seeing a very high failure rate of our samples.
Thanks
Andrew
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