Sorry - our cuvette temp is 50oC. Don't know why I typed 35oC!
-----Original Message-----
From: Walding Andrew
Sent: 25 August 2000 09:54
To: Recipients of ABRF List
Subject: RE: Injection on the 3700
I have several replies to both of my recent queries. The latest run we have
done (unfortunately) means that I can't see a solution yet!
1. We have just had the needles replaced. I suppose they could be causing
the problem, but I think this is unlikely.
2. The 'load bar' has been replaced by AB about 2 weeks ago. Is that the
'board' that Martin mentioned?
3. Our sample quality is fine. We have always been extremely careful with
sample preparation - isopropanol precipitating every DNA prep (and then gel
quantitation); EDTA precipitating our sequence reactions; resuspending in
water or 0.5mM EDTA (10-15ul).
4. Our injection is 1000V, 30 seconds. I would like to optimise this. An
AB engineer has been in and optimised our cuvette temp in the past to 35oC
(but about 3-4 months ago). Perhaps we should redo this? Does 35oC seem
too low?
5. We did two spatial runs yesterday and the day before, and they all
passed, and looked fine (although the array view disagreed with the spatial
calibration 'chart'...)
6. Yesterday we ran the AB 3700 standards (add water and run...) in every
other capillary (Rows C, D, G & H). The array view showed every other
capillary to have a strong signal, and the others to be empty (black). When
we analysed the data, we found:
(a) The data from the loaded capillaries varied massively (from 0 to
700 bases) - mostly poor.
(b) We got up to 400 bases of semi-decent quality data from
capillaries that had NO sample in. These aligned nicely with the other
lanes! For example, well B3 gave 537 bases of the control sequence. I've
just looked at the signal strength - less than 10!! But I shouldn't have to
check all of my files to see what the signal strength is!!
I have image files if anyone wants to see them!
This is a long email (sorry!). I hope I don't send it twice this time!!
Thanks for all your advice so far! If you can still help, I would be very
grateful!! We are getting very frustrated here (should have got a Megabace,
perhaps...)
Andrew
-----Original Message-----
From: Cairo, Al [mailto:Al.Cairo@pfizer.com]
Sent: 25 August 2000 00:39
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
-------------------------------------------------------------
Alfred Cairo
Pfizer Global Research & Development
Alameda Laboratories
1501 Harbor Bay Parkway
Alameda CA 94502
phone: 510-749-6220
fax: 510-523-0886
-----Original Message-----
From: Bayliss, Stuart [mailto:stuart.bayliss@csc.mrc.ac.uk]
Sent: Thursday, August 24, 2000 8:56 AM
To: Recipients of ABRF List
Cc: abrf@aecom.yu.edu
Subject: RE: Injection on the 3700
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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