"Thomas J. Stelick" wrote:
>
> Hello from the frozen north,
> I was wondering if there was a consensus on what is the best
> way to load the 96 lane gels? I use a modified water protocol and it
> seems to work fine, but there probably is a better way. What are
> people using to get the best reads. We use the .4mm abi comb. thank
> for the info.
> Tom Stelick
> BioResource Center, Cornell University
> 157 biotech bldg.
> Ithaca, NY 14853
> (607)254-4857
> tjs11@cornell.edu
> http://brcweb.bio.cornell.edu
Hello everyone.
I am happy to finally be able to contribute to the group rather than
just lurking in the background collecting info from all of you. I thank
you all for all I have learned from all your experiences.
In our lab we use membrane combs, and regular plates (not the troughed
plates) with 0.2 mm spacers. Our protocol doesn't rely on the water
protocol, so in my humble opinion it's easier to do and much easier to
teach to new people. Because we aren't using the waer load method,
there is no buffer tank to look through, or water to load through, and
no mixing of water and buffer prior to running. We have also noticed
that we don't get the compression and distortion that are sometimes
common to the water load protocol.
Something else we noticed is that we don't have to worry about stagger
loading our samples, as the membrane combs provide sufficient dark space
between the lanes for the tracker to work properly. Consequently, we can
load all 96 samples at the same time after dipping the membrane comb
into a loader tray that has the resuspended samples in small wells.
Our protocol uses no gel prerun (only a short gel pre-warm step) so it
doesn't take very long to complete. We can load 96 samples on a 0.2 mm
gel in under 15 minutes--starting from the time you actually finish your
plate check to walking away from the instrument and letting it go
through the collection. I've tried this method with dRhodamine and
BigDye chemistries, on PCR products, plasmids, ESTs... and so far this
system has worked well for all of them. PHRED and Factura scores
confirm our sequence quality and read length to be as good or better
than any other method we have tried.
I am currently trying to write this up, but in the meantime I would be
happy to send you a protocol if you are interested.
Cheers,
Derek
-- .-. .-. .-.Derek M. Harkins <derek_harkins@ncsu.edu>.-. .-. .-. /|||X|||\ /|||X|| North Carolina State University ||X|||\ /|||X|||\ X|||/ \|||X|||/ \||| Forest Biotechnology Group X|||/ \|||X|||/ \|||X `-' `-' `-' Raleigh, NC 27695 USA (919) 515-7800±1 `-' `-' `-'
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