ABI 373A automated sequencer - logistics of use/experience

John H McDonald mcdonald at ravel.udel.edu
Fri Sep 3 19:06:32 EST 1993


In article <3SEP199315084174 at aardvark.ucs.uoknor.edu> broe at aardvark.ucs.uoknor.edu (Bruce Roe) writes:
>In article <1993Sep2.131659.29560 at gserv1.dl.ac.uk>, daj at uk.ac.ic.nhm ((David Johnston) daj) writes...
>>Greetings,
>>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

For someone who is already familiar with 'old-fashioned' sequencing, a
couple days training should be enough, in my experience.  

>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
>costs.

Actually, the latest pricesheet I have lists them at $530 a set.  They're
made of "optically flat, low fluorescence Vycor glass from Corning," 
according to a paper in Gene Analysis Techniques and Applications 9:9-16
(1992).  Has anyone looked into buying some of this glass and having the
local glass shop make a few plates?

>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).

The Ericomp Twinblock and Powerblock cyclers with heated lids also don't
require oil (definitely a plus when doing the small-volume sequencing
reactions), and cost about half as much as a Perkin-Elmer 9600.  The Idaho
Technologies Air Thermal Cycler, which does the reactions in capillary
tubes, is very fast, doesn't require oil, and also costs about half as
much as the PE 9600.  Despite initial worries about the halogen lamp in
the Idaho machine fading the dyes, I'm getting excellent sequence using
one.  You just have to remember to add some BSA to the sequencing reaction
to keep the taq from sticking to the glass.  Standard disclaimers, of course.


>
>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.

You can reduce costs by doing 10 ul reactions instead of 20 ul, keeping
the concentrations of everything the same.  There is still plenty of
signal (at least with dye terminators--I've never done dye primers).
I suspect you can also reduce the concentrations, and I'll be doing some
experiments soon; anyone out there know how little of the expensive dye
terminators we can get away with?

>
>>
>>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.

I'm using 128 Mb magneto-optical drives for storage.  The drives are under
$1000 and the disks are about $40 each.  This way everyone in the lab can
be responsible for their own data, since each disk will hold about 1000
analyzed sequences.

>>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.
>


All I've ever done is Taq cycle sequencing of PCR products.  It works fine
for me.  The keys are getting enough template, and getting clean template.
 I always gel purify (by spinning the gel chunk through filter paper). 
A 'typical' 100 ul PCR reaction gives enough template for six or more
sequencing reactions.  Also, I always sequence both strands.  There are
generally a couple of ambiguous bases per sequence, but the ambiguous bases
are always in different positions on the different strands, so it's not a
problem.

John H. McDonald
Life and Health Sciences
University of Delaware
Newark, Delaware  USA



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