In article <502am2$dqj at net.bio.net>, Sensational Gravity Boy
<ed at bio02.bio.uottawa.ca> wrote:
> Hi all, sorry if this appears twice, but I don't know if my first post is
> lost in the lines. Here is my problem.
>> We ran a gel last night (on a 373) and although the gel file looks fine,
> the samples have a spacing of -12 !!! yes, minus 12. As a result, the
> chromatograms look horrid and there are stacked bases that are
> not been called, and essentially the 30 samples we ran are a total waste.
> Again, on looking at the gel file, everything looked normal so we're a
> bit shocked. Has this happend to anyone before ? we were using a new
> batch of polyacrylamide, but I doubt this would be the cause of the
> problem. Is it an analysis problem ? Is there a way to extract useful
> data from what appears to be a fine looking gel ? Anyone ever have such a
> weird spacing ? We've had problems before, but due to buffer problems,
> and these led to strange spacing calls, but the batch of buffer used on last
> night's gel has been performing well. We're puzzled and bewildered.
>> thanks for any and all ideas.
>> Ed Taboada
> Dept of Biology
> University of Ottawa
> Ottawa, Canada
>>> PS. please hurry !!! :) we're going to run a gel with 4 samples that
> have sequenced well before as a test. But we'd hate to destroy a gelfile
> that may have had analyzeable data (the space left on the hard drive of
> the said computer would not allow us to keep both gel files).
>> thanks again.
Ed,
We have seen the -12 base spacing problem with a good looking gel image
occasionally. We have found that it does correlate to a bad batch of
acrylamide. We have traced it to the shipment of acrylamide being heated
above four degrees for extended periods of time during the shipping
process. (We use burst-pak gels in which the acrylamide is already made
up...if you made your acrylamide from powder, maybe check the temp of your
refridgerator???)
In any case, when we got new acrylamide, all of the problems went away.
We have found that with a 373 run using the ABI50 Basecaller, that
reanalyzing the samples with the semi-adaptive base caller helps a little,
but if you have a problem with your acrylamide, its faster in the long run
to just send it back and get a new batch.
Michele Godlevski
Glaxo Wellcome Sequencing Facility
Research Triangle Park, North Carolina
