Wendy Fuller asked.>
>I am interested in communicating with people who are routinely using or
>>attempting to use the Perkin-Elmer 96 lane upgrade. We have been working
>>with the upgrade since October and are still having problems; namely, how
>>to flush and load the wells. We have tried several methods but even the
>>most promising one appears to cause leaking of the wells which in turn
>>causes the tracking to be a total nightmare!!
>>>>I am also interested in speaking with people who are ROUTINELY using the 48
>>and/or 64 lane upgrades as well.
>>>>What are your opinions and experiences regarding these extra lane upgrades
>>(48, 64, and 96)?
Our response>>
In general I would say we are very satisfied with the upgrades.
>Here at Iowa State we do custom DNA sequencing which means we may use 4
>or 5 thermocyclers for a set of reactions to accomodate various cycling
>conditions. Some samples may be annealed at 60 C or 45 C, or we may
>denature for a longer time. Because we do custom sequencing, our
>situation does not lend itself to using 96 well format and multichannel
>loaders.
We have settled on using 80 well combs from The Gel Company. This was an
idea of Laura Livingstone at UNC Chapel Hill. The 96 lane upgrade allows
80 lanes of the well width of the 64 lane comb. The tracking can be a bit
of a problem because the software expects the lanes to be narrower, so if
there is a series of lanes which do not work, extra lanes wil be inserted
in the blank area. Easy enough to correct and it does take some time, but
it does allow 80 lanes per gel. Loading 80 lanes one at a time is a bit
tedious but it does work.
We found that flat tips from Dot Scientific Inc. (800 878-1728) Cat. No.
RN015f-LR2 were consistantly narrower and fit the width of the 64 or 80
lane combs very well.
We found that the Gel Company (415 24-8760) combs Cat. No. CAK80U-020 were
more uniform in thickness and better matched to the Gel Company spacers
than the combs and spacers used before. Unfortunately nothing is perfect.
Sometimes the comb used to cast the gel will not fit between the plates. I
just routinely grab 3 or 4 combs from our supply and find one that fits
tightly. If the comb fits tightly, there should not be leakage sideways
between lanes. Most of the time our leakage is from sample overflowing the
well into the next lane. This is most noticeable when a sample with very
high signal gets into a lane with weak signal. If you get some of the
Amersham pink loading buffer, you will see very quickly whether the leakage
is between lanes or from poor placement of the sample into the intended
well. We routinely use the blue dextran but I found that a little extra
blue dextran in the loading buffer makes it easier to see the loading
process. If you notice some overflow into an adjoining lanes, this can be
gently flushed out with loading buffer.
The gel company also makes 96 lane combs which are 0.2 mm and is used with
the standard plates not the one with the ground surface. Bruce Roe's lab
uses them and loads multichannel.
I think if people would stop expecting everything to work perfectly
everytime, then some of their problems would become less of a problem.
Now if you want to to risk gel extrusion, you can spray silicone on the
combs and they will not leak sideways. Wide any excess before using the
comb. You must be careful to get rid of the silicone on the plates
because they will extrude. Guaranteed!!!!! If you have the Labconco
dishwasher, then the silicone probably won't cause total disaster. Not
really recommended to use the silicone.
Hal
Harold G. Hills, Ph.D. DNA Sequencing Specialist 515 294-9585
1184 Molecular Biology Building FAX 515 294-1597
Iowa State University hhills at iastate.edu
Ames, IA 50011-3260
http://www.biotech.iastate.edu/Facilities/DSSF/ABRF For ABRF 97 DNA
sequencing tutorial.
http://www.biotech.iastate.edu/Facilities/DSSF For info about facility.
