Hi there,
I have had similar problems although the circumstances were different. If
the size standard for the fragment analysis in GeneScan is dsDNA (like
e.g.
GS-350 or GS-500) or ssDNA (like GS-2500) cabable of making secondary
structures due to a cold start, they will migrate differently. Cure:
prerun
for at least 1 - 1,5 hours or make your own size standard with DNA that in
GC content, size, and composision are more comparable to your unknown DNA.
M., Q., BARI, bqamar at paknet1.ptc.pk wrote:
> Hi!
>> I need a little help with the ABI 377 and GeneScan analysis. These days
> I am trying to run more then one GeneScan gel per day, the gel that I
> run in the morning runs fine but then when I try to run another gel on
> the same set of plates, after washing them, I start getting problems.
> It seems that the migration of the fragments on the second gel is
> invariably slower and so the size of the fragments that I get is greater
> on the second gel. This is without changing anything else including the
> buffer and the acrylamide. I am stymied and do not understand what I am
> doing wrong or what to do? It seems like the sequencer gets tired by
> the end of the day so the migration anomaly ;-) If anyone has
> experienced a similar problem and found out how to solve it please let
> me know as I am pressed for time and have to get a lot of data out as
> soon as possible.
>> BTW I have encountered this problem a number of times and now I have
> made a standards file based on this "Bad gel" and analyse my samples on
> these slower migrating gels on the basis of this set of standards file,
> is this valid to do? I have tried to re-run and compare the size of the
> alleles on the slower and the normally running gels and find that: for
> the smaller fragments the sizes are the same. However, for the larger
> fragments the sizes usually do not compare between the gels.
>> My instrument is relatively new so it could not be that the electrodes
> have corroded.
>> Hoping to hear from you all.
>> Raheel Qamar
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