We are interested in purchasing automated sequencer
woconnel at nmh.org
Wed Nov 12 05:54:06 EST 1997
We have had our two ALFexpress machines only since June and we were able to
do heterozygous detection within a month. We chose the Pharmacia machine
because we felt it was better at this application than what was then on the
market (we only compared the ABI and Pharmacia sequencers). We now use the
Mutation Analyzer software for heterozygous detection analysis and find it
very easy to use after you get the parameters set up.
We also use Amersham's Thermosequenase kit almost exclusively, but haven't
switched over to the Amresco PAGE-Plus but plan to as soon as the long
ranger is used up. We are sequencing pcr products of about 400-500 bp long
so we are able to get away with shorter runs and are able to do multiple
runs in a day.
The software issue at times is a pain in that most of the software written
for sequencing is in a Mac format most notably Sequencher.
I don't find that clean up is difficult. The plates are relatively small
and I am as careful cleaning these plates as I am with any other sequencing
I am very excited about the merger of Amersham and Pharmacia. Not only does
Amersham make the Thermosequenase kits and have extensive experience with
sequencing (USB and Sequenase) but it also has the license for the Cy5 label
which the pharmacia system utilizes. One can only guess at what may come
out on the market with Amersham optimizing kits for the ALFexpress (vs.. the
ABI systems). Also since there is only one label (versus 4 for ABI) it is
much cheaper to make labeled primers.
Lastly I readily agree with Philip that the customer service has been
terrific. Marc Goldstein, the Application Specialist, has been
indispensable and has shown great enthusiasm in getting our projects off the
If you have any specific questions about heterozygote detection with the
mutation Analyzer software and the ALFexpress don't hesitate to get in touch
with me at woconnel at nmh.org
Molecular Genetics Lab
Northwestern Memorial Hospital
250 East Superior Street
Chicago, IL 60611
woconnel at nmh.org
Phillip San Miguel wrote in message <64a4s2$p4c at net.bio.net>...
> We've had an Alf express for over a year now. Over all a good, robust
>machine that with some (perhaps extensive) optimization will give 800 bp +
>average, >99% accuracy) reads. Its easy to pour gels, easy to load (the
>wells are backlit) -- clean up is not too bad. I would warn you to use
>thermosequenase, not the pharmacia kits --
>but as Pharmacia and Amersham have merged, your service rep. may give you
>the same advice.
> Throughput is not that high -- 10 samples/gel. But if you really push
>it, you can get 2 gels run a day (one per 12 hrs) and still get reads over
>900 bases. We've only been able to do this since we switched from Long
>Ranger to AMERESCO's PAGE-PLUS acrylamide. [By the way, remember when you
>are asking about read length to ask for read length at a certain accuracy.
>Alfx and LiCor's can potentially read >1000 bases at >99% accuracy
>(ambiguities don't count). On a decent Alfx gel I get one read over 1000
>and average in the high 800's.] You could get higher throughput if you
>just need short reads -- there are short plates that run very fast -- but
>I've never used them.
> The software is quite good -- it runs in Windows 95. There are a few
>irritating things about it, but nothing worth mentioning.
> Okay, now the down side. You can't load the wells with a 8 or 12x
>hamilton shringe loader -- at least not one that will pull up from a
>standard 96 well plate. This is pretty minor because it doesn't take that
>long to load 40 samples (4x10) -- maybe 1/2 hour with a little practice --
>maybe faster. The plates are pretty expensive, $900 for the thermoplate,
>$150 for the front plate. Don't break them. I worry about the durability
>of the theromplate as it is a pretty complex thing. But the Alfx's worst
>problem is its "clone 10" problem. The samples are numbered 1acgt,2acgt,
>etc.; from the left (close to the laser source) to the right. (I should
>add that this is a problem you won't even notice unless you get everything
>optimized so you reads are averaging over 800 bases and are pretty even.)
>Each of your samples (acgt) is called a clone. The first 8 clones are
>capaple of delivering reads over 900 at >99% accuracy routinely. I don't
>remember seeing clone 9 break 900 bases and clone 10 never (in any of the
>120+ gels I've run) breaks 800 bases. Again, unless your willing to put a
>lot of effort into optimising your sequencing you may never even notice
> Finally, I highly recommend Pharmacia for their service and sales
>representatives -- at least the ones we deal with, Marc Goldstein and John
>Paul G. Rothberg wrote:
>> We are considering Visible Genetics vs. Pharmacia automated sequencers.
>> Input would be greatly appreciated from users. Send to
>> JBRADLEY at CCTR.UMKC.EDU
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