Agnes Gallacher wrote:
>> Hi,
> Has anyone tried using 48cm plates for a 96 lane run? If so, how did you
> go about it and what were the results like? Was it worth the effort?
>> Agnes Gallacher.
>
We use 96 lanes (etched plates) with 48 cm WTR. We get reads of about
850 usable bases in 8-9 hours using amresco, fmc or sigma gel mixes
(running at 2500 volts), no water loading, using a kloen loader (loading
from the bottom of the well up). Our phred q>20 scores range from
400-550 using dsDNA and bigDye terminators. They increase to almost 600
using M13 and BD primers (we don't run M13 or primers very much anymore
though). The 36 cm WTR plates give shorter usable reads. 48 or 64 lane
gel are either not high enough throughput or disrupt the loading patter
from 96 well plates.
The new software (both 3.2 or 3.3) works well for tracking if you have a
high pass rate for your sequencing reactions, but if you have failures,
it can be a problem. We actually run matrix stds in lane 1, 49 and 98
and then have to move three lanes to take this into account, but it
makes tracking a lot easier and you don't lose any lanes.
Bottom line (for us) is that we give up a little quality for increased
throughput. We will probably not be investing in plate based
sequencers anymore, and going with capillary sequencers in the future.
hope that helps
srlasky
--
Stephen R. Lasky, Ph.D. #
University of Washington #
Department of Molecular Biotechnology #
srlasky at u.washington.edu #
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