NEW Sequencing Gel Problem

Andre Hamel hamel at ccu.umanitoba.ca
Tue Mar 2 18:34:02 EST 1993


In article <9303021634.AA11832 at map.marc.usda.gov> hawkins at MAP.MARC.USDA.GOV (Greg Hawkins) writes:
>
>        Our lab needs some helpful explanations for our new sequencing gel
>problem.  We are very experienced with sequencing using the Sequenase kit
>and AT-Biochem's Long Ranger gel (5%).  Our problem: When we run our gels
>for 1.5 hr (short run) our sequencing is perfect.  On the SAME gel we run
>the same samples for 3 hr (long run) and get smears (no banding pattern at
>all).  We control the temperature of the gels carefully.  We have run
>hundreds of samples like this in the far and recent past with no problem.
>
>        Our conditions:
>               - M13 
>               - Sequenase kit
>               - NEN S-35 dATP
>               - 40 cm gel; 5% AT-Biochem Long Ranger
>               - 1 X TBE
>               - 55 Watts
>
>        Is ther something wrong with this batch of Long Ranger??  Could it
>be a running buffer problem??  Please respond soon!
>
>                Thanks!
>
>                hawkins at map.marc.usda.gov (Greg Hawkins)
>


How's your TBE stock? Is it stored in glass or plastic? Even if no
precipitate is evident (especially if in plastic), autoclaving wouldn't
hurt before making up 1x working solution. Also, buffer depletion can
occur for longer runs if buffer reservoir is not large enough (say 500 mL
or less for each anode and cathode).

Also, how wide are your smears? Same width as the well? If so, then
perhaps templates have too much RNA in them (alk. denaturation of at least
5 ug template in  0.2 N NaOH for 15 min. at 45oC, followed by addition of
0.3 M final con. NaOAc, pH 5, then 4x vol. -20oC ethanol, then -70oC for
20 min, ufuge top speed 10 min, wash pellet in -20oC 80% ethanol, ufuge
again, dry, etc.

Andre Hamel
Manitoba Vet. Virol.
Winnipeg, Manitoba (where else do you think Winnie the Pooh bear get his name
from?)
Canada

email: hamel at ccu.umanitoba.ca

TTFN



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