ABI 373A automated sequencer - logistics of use/experience
broe at aardvark.ucs.uoknor.edu
Fri Sep 3 15:08:00 EST 1993
In article <1993Sep2.131659.29560 at gserv1.dl.ac.uk>, daj at uk.ac.ic.nhm ((David Johnston) daj) writes...
>We have just secured funding for an ABI 373A automated sequencer (a very
>big thank you to the Wellcome Trust). I know that ABI provide training in
>the actual operation of the machine. However, as our system is to be
>"shared" between several projects run by different Departments in the
>Museum, I am interested in the wider issues affecting the day-to-day
>running of an automated sequencing facility (the practicalities and
>logistics) and in trying to anticipate potential problems. For example;
>is multi-user operation practical? If so by how many users? Or is
>dedicated technical support essential?
A multi-user operation is NOT practical unless you or someone has
extremely tight control over them, i.e. in my lab we have 4 ABI's
and each of my students take turns (after a month or 2 of training)
running the 373A's. If you have a single ABI 373A, then I STRONGLY
recommend dedicated technical support!! Part of the reasoning behind
this recommendation is that cleaning and pouring plates takes lots
of practice, actually loading the gel is no problem BUT making sure
the plates are clean is. Also, it seems to take some skill in handling
these plates (remember they are $1000 US/set) so break one and it really
I also STRONGLY recommend that each gel run contains a STANDARD
that YOU KNOW works and a second STANDARD template reaction run by
the individual who gives you their samples. The biggest problem is
garbarge templates prepared by folks who don't really know what
they are doing. You should create a protocol for ss and ds template
preparations and get everyone to use it or else no guarantees that
the data will be reasonable.
Since you've got the money for the 373A, also buy a PECetus 9600
for the cycle sequencing reactions. NO OIL required and very reproducable
temperature cycling (disclamer: I don't work for them or have any personal
gain, just a very satisified user).
Lastly, remember "simplest is best", get ss (m13) templates to work first
and buy the kit from ABI, then get ds (pUC or other plasmid) templates to
work second, again using the ABI kit. Then try the terminator kits from
ABI with the above template preparations that you know work well with
the fluorescent-labeled primers. Once all that's been done and standardized,
then give PCR-product based sequencing templates a try. Again, the philosophy
here is to try the simplest templates first and then move on to templates
that are more difficult to isolate at a sequence quality purity.
Note on kits: I hate kits and usually don't advocate buying them BUT
ABI has a vested interest in getting their instruments to work and work
well and is the sole source for these kits which do work quite well.
You can make your own reagents, but for a site with on 373A, the bother
is not worth it.
>Prefered specification of networked Macs, VDU size, RAM, Disk size etc.
While you've got the money, buy a Sun computer with 2 gig hard disk
and get the MRC/WashU programs XBAP and TED. Alternatively, Sequencher
and DNAStar might provide you what you need and are Mac-based.
>Raw data backup policies.
We back up nightly.
>Storability of sequencing reactions.
The cycle sequencing reactions can be stored frozen immediately after
the reactions are cycled for several days without noticable change.
Just take the tube rack out of the PECetus 9600 and put it directly
into a -20 deg. freezer. BUT once you pool and add the loading mix, forget
storing them. They go off noticable in 24 hours if frozen with the
loading mix (don't really know why but they do).
>Prefered chemistries; Taq vs Sequenase, dye primer vs dye terminator
See above. Simplest is Taq cycle sequencing with dye primers on ss
templates. Our experiences with the new 5 filter wheel and the Sequenase
terminators is excellent. Much better than with the Taq terminators.
>Level of maintenance cover
Get the FULL service contract. Our laser dies at least once a year
and the cost of that alone is worth the FULL (I think they call it GOLD)
service contract. We get prompt, excellent service from ABI when one
of the 373A's goes to hyperspace.
Getting good template. Getting people to RTFM (read the #%$^& manual).
>If anyone has any comments on any of the above (or on anything else I have
>forgotten), then I would be most grateful to hear from you.
>Similarly, if anyone running a busy 373A facility (genome project?) reads
>this and would be prepared to have me look over their shoulder for a day or
>so, please get in touch.
You are welcome anytime in Oklahoma, just stop by Cambridge and pick up
some Abbot ale. A couple of liters for each day you visit. Seriously,
the problems are 99.999% what you do before you load the samples, i.e
sample preparation, so don't think you'll have any reason to visit other
users for the actual operation of the instrument after the installation
and brief training.
>Thank you, in advance,
>David A. Johnston
>Dept of Zoology, The Natural History Museum, Cromwell Road,
>South Kensington, London SW7 5DB.
>(tel 071 9389297, fax 071 9388754, email daj at nhm.ic.ac.uk)
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