Two weeks ago we switched from the 48 to the 64-lane shark tooth comb at
our DNA sequencing on ABI 377. Albeit everything (except of the comb)
remained identical in comparison to prior conditions (even the sample
amount which is 1=B5l), our results are remarkably worse. We receive a
significanty higher background especially towards the end of the run and
have problems with diffusion (or shining) of the samples into (upon) the
neighbouring lanes. Additionally, we are under the impression that the
software has more problems to handle weak signal lanes at 64 lanes than
with 48 lanes. Is this true or is it just an imagination? We are currentl=
y
using NaOAc/ethanol precipitation for the workup of the sequencing reacti=
on.
Do you have any suggestions to overcome these problems? What is the
"normally" used loading volume for the 64-lane comb (I know that the
96-lane comb is used with 0.5 =B5l, do you think reducing the sample volu=
me
could help?)?
Any suggestions are highly appreciated. Thanks in advance.
Yours,
Marcus Macht
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Marcus Macht
ZMMK-Servicelabor
Joseph-Stelzmann-Str. 52
50931 Koeln
Tel.: +49 221 478-6995
Fax: +49 221 478-6977
e-mail:Marcus.Macht@uni-koeln.de
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