Dear Fellow Sequencers,
A couple of people have e-mailed me today, to ask what gel oozing is.
There follows a summary and description of the problem. There have been
two major calls for help with feedback summaries posted to the group by
Harald Gills and myself. Find these by searching the newsgroup archives,
for example at: http://www.deja.com/.
Gel oozing, more correctly (?) termed gel extrusion and once referred to as
gel creeping, is the physical expansion of the gel itself, during a pre-run
or a run. It isn't a feature identified on the gel image. It appears to
be only associated with the ABI 377 family, although I cannot qualify this.
Gel extrusion can be fatal to a run: I've seen the gel expand so much that
it has come out from between the plates. More common is that the gel
extrudes (expands upwards) so that the wells become severely distorted, and
un-usable. A good short gel can be used <3 times for genotyping, so gel
extrusion makes runs 3X more expensive for us, and more time consuming.
Sometimes, the wells become distorted during a pre-run, affecting the whole
run. Other times, the gel extrusion only comes after the samples have been
electrophoresed into the gel and the effects are less severe to the run in
progress.
The phenomenon is seen around the world in different labs. Different
people have different experiences (e.g. we find the incidence og gel
extrusion rises significantly during hot humid days. Also, some people
(including myself) believe that we see less frequent and less severe
extrusion, when using Long Ranger instead of acrylamide. Other members of
my lab don't have the same belief - we are about 50/50 as acrylamide/LR
users).
The phenomenon is very frustrating for any lab that has a series of gel
extrusions, not to mention the cost. It's a real pain when sequencing.
There are no "cures" that I know of, or that have been published here, to
my knowledge. People have tried using fresh batches of enzymes, leaving
the gel to polymerise longer, heat treating the plates, cleaning the plates
with acids or alkali, dishwashing the plates, running at lower temperature
et cetera.
Each lab has their own experience with the success of their chosen
treatment, but there is a general agreement that we don't *know* what is
causing the extrusion phenomenon, and therefore don't know how to banish it
once and for all.
Personally, I think that the most common treatment that appears to be
successful is baking the plates at 70 degrees C for 2 hours when extrusion
appears. The forensics lab in the same building as us do not have a
problem with extrusion, even though we use the same gel recipies and
reagents. They do, however, wash their plates in a dishwasher. I would
like to see the heat-treatment tested in as many labs as possible, who
experience gel extrusion, to see if we can establish whether or not this is
a cure. Feedback of this nature should be mailed to me directly.
I hope this helps!!
Cheers,
Andrew
--
Institute of Immunology
The National Hospital University of Oslo
Oslo, Norway
Author: http://www.brunel.ac.uk/depts/bl/project/biocomp/sequence/seqanal_guide/
>Dear Andrew,
>>sorry for my stupid question - but I dont know what exactly you mean
>with "gel oozing". Is there a picture of such a gel on the Web? Can
>you quickly define what you mean with the term? We had trouble on our
>377s some time ago, but I can not tell if the problem we had is "gel
>oozing".
>>Thanks and best regards, Bernd
>>>>>>After reviewing the many replies about gel oozing, I came to pretty much
>>the same conclusions as you - except that you didn't mention heating the
>>gels. I cannot offer any suggestions as to how this might work (offers?),
>>but it was clear that the labs which used a dish washer (which has a high
>>heat drying process at the end) or the labs which heat treat their plates
>>(2 hours at 70 degrees C) when gel oozing appears, have no problems - or at
>>least markedly fewer problems.
>>>>It would be interesting to hear if other labs find this works. If you do
>>heat treat your plates after an oozing incidence, please let me know if it
>>works. Let me know if it doesn't too, please!!. If I get some responses,
>>I'll summarise them to the group.
>>>>I was very interested to read your theories about the differences between
>>373 and 377 runs. I'd like to add that several of us notice less oozing
>>with Long Ranger gel rather than acrylamide. Of particular interest was
>>your comb theory. One of our researchers realised a few weeks ago, that we
>>too had been using water to support the wells when removing the comb, and
>>then later loading the buffer tray with TBE. We now have a squeezy bottle
>>with TBE instead of water, to hand. Unfortunately (!) we haven't had any
>>problems for a while, so I cannot say if that is the cause, but this may be
>>more practical than absorbing the water with a paper towel?!
>>>>I wish you luck on your crusade, and hope that you can help to solve
>>the riddle.
>>>>Kind Regards,
>>>>Andrew Louka
>>>>--
>>Institute of Immunology
>>The National Hospital
>>University of Oslo, Norway
>>>>>>>>>> Sent via Deja.com http://www.deja.com/>> Before you buy.
>>_____________________________________________________________________
>>PD Dr. Bernd Weisshaar
>MPI fuer Zuechtungsforschung
>Carl-von-Linné-Weg 10
>50829 Koeln
>Germany
>>Tel. office: +49-221-5062 590 Tel. lab: +49-221-5062 311
>Fax: +49-221-5062 313
>mailto:weisshaa at mpiz-koeln.mpg.de>WWW-URL: http://www.mpiz-koeln.mpg.de/~weisshaa/BW-info.html
Sent via Deja.com http://www.deja.com/
Before you buy.
