IUBio Biosequences .. Software .. Molbio soft .. Network News .. FTP

250-300 dip

Alan Wilton dna at unsw.EDU.AU
Wed Mar 25 10:34:38 EST 1998


Dear Hal, Emma and whoever is interested

I do not believe it is the buffer.
Everyone I talk to who is running 377s seems to be having
this problem to some extent and I think it is a build up
on the plates.  If we scrub the plates manually very
vigorously during 3 or 4 alcohol washes and the polish
the problem goes away (even though using the same TBE buffer)

A previous posting on the subject is below.

Alan Wilton

>Note to Emma Puttick.
>
>Could not mail you directly.  Check out the ABRF tutorial at URL below for
>the item under Taurine buffer.
>
>Hal
>>
>>We have been experiencing a consistent loss of peak height intensity and
>>resolution in 30bases in the 250-300bp region. We see this on every gel
>>and in every sample. After that region the intensity and resolution
>>improves and we can usually read out to 600-700bp. We have seven 377's,
>>which we run 2-3 times/day doing 4 hour runs with 29:1 gels and we see
>>it on every gel and it doesn't matter which chemistry we are running.
>>This week we will be doing a series of experiments, on the reverse side
>>of the plates, which will hopefully isolate the reagent(s) which are
>>causing this problem of build-up on our plates. Our second problem is
>>restoring the original side of the plates.
>>I have heard that MultiTerge is meant to fix this problem, but we cannot
>>buy it in Australia, yet.
>>
>>Does anyone have any ideas on what is causing this effect and how to
>>eliminate it? Are there alternatives to MulitTerge?
>>
>>Any sugeestions are very much appreciated!
>>
>>Thank-you,
>>
>>Emma Puttick
>>DNA Sequencing Supervisor
>

Dear Sequencers and Genescanners,

There have been several requests for assistance with gel problems on
ABI377s recently on the newsgroup.  We have recently come through a very
trying time with similar problems and here is a solution that works for
us.

PROBLEM
Something is sticking to the plates that affects the migration of the DNA.
It causes a loss of resolution, stretching and retardation of bands over
150 basepairs.  On slightly affected plates the sequence will come good
after 300 basepairs.  The distortion is affected by the concentration of
DNA, more DNA less distortion.

RESULT
Sequencing gels that only give 200 bp even with strong signal.
Genescan gels with standards over 150 bp missing or reduced signal for 250
and 300 bp standards.  Genescan gels with 2 sets of peaks in the same lane
for standards over 150 bp.  They differ by about 2 bp.

SOLUTION
Clean them initially by soaking them at least 20 min in 2M NaOH
and then rinsing in 2N HCl for 10 min to neutralise them.
Then rinse them thoroughly in water and wash them well with alconox.

We also routinely soak plates in NaOH for 5 min and rinse in HCl.
We have to also wash 3 or 4 times in ethanol scubbing each time very hard with
lint-free tissues then polish the plates with tissues using lots of elbow
grease.  We have to do this procedure everytime we run a gel or the
problem returns.

If anyone knows what the cause is we would like to know.  Prevention is
better than cure.  We do not touch plates with gloves because that was a
suspected cause.  I thought it might have been the Sydney water but it
seems to be a world-wide problem.  I wonder if it is the Alconox?????

Best of luck all,

Alan Wilton

>Once in a while we experience partial loss of peak intensity at about 2000
>points while sequencing with ABI 377.  Sequences start very nicely,
>diminish around 2000-2500 points and come back very nicely afterward.  Any
>suggestion?

----------------------------------------------
Alan Wilton
School of Biochemistry and Molecular Genetics
University of New South Wales
Sydney  NSW  2052
Australia

Phone	+61 (2) 9385 2019
Fax   	+61 (2) 9385 1483
Email 	a.wilton at unsw.edu.au





More information about the Autoseq mailing list

Send comments to us at biosci-help [At] net.bio.net