Thank you all very much for your responses to my question on weekend gel
storage. You have been extremely helpful.
My question:
We are using the Perkin-Elmer ABI 377 Prism for mapping. We would
like to make our gels in advance, Friday in preparation for early Monday
morning. We have tried to store them over the weekend and have found
they dry out even when the top and bottom of the gel are wrapped with
plastic wrap. We think that perhaps we can prevent this by wrapping the
gel and storing it in the refrigerator. Have you had any success with this
method, or do you have any suggestions for us?
Thank you very much for your help.
Summary of the responses:
1. Don't try it. The urea will precipitate. Get someone to come in early to
pour the gels and use them after 1.5 hours. For genescan it should be ok
but not for sequencing.
2. My general policy is not to use any gel that is more than 4-6 hours old for
sequencing because of loss of resolution beyond 550 bases. Our genescan
gels we do pour late at night for the next day. We have a 3-midnight
shift.
3. I don't have the 377 yet (on order) but I've found that gels for the 373 do
store well when wrapped in cling film/saran wrap with a wetted tissue
inside (maintain humidity) at 4C (ie. fridge or cold room).
4. I have left gels out overnight (controlled expt.'onest) on the bench and
found them OK for a run the next day.
We store our gel also over w/e and we plastic wrap them but we put
between a paper tissue at both sides of the gel with running buffer and
no problems.
I wouldn't put the gel in the refrigerator because the urea will
crystallize.
5. Saturate paper towels with 1X TBE and then wrap the gels with plastic wrap.
6. Have you tried aluminum foil? Place several damp paper towels around the
plate and then wrap the entire plate in foil. Our lab has been able to
use plates stored at room temperature for several days after pouring. I
don't know if I would recommend refrigerating them. Since the the gel is
so thin, this might cause more damage to the gel.
7. You might try wetting some towels with TBE and putting these in the
saran wrap
with the gel. HOWEVER- Gels can break down over time when stored and although
GeneScan gels are more forgiving than sequencing gels in many ways the
resolution is still dependent on gel quality. Because the time spent
processing
the data increases dramatically as data quality decreases it is vital to test
the run to run precision of your gels empirically to avoid costly delays.
8. I know of many users who pour gels 24 hours in advance but am unaware of
anyone
who keeps gels longer. Hopefully other replys to your message will be
informative in this regard.
9. We thought of this for convenience when we first started, the
advice we were given was that, for the gels still to be accurate then the
run that they were going to be used for, should be finished within 24 hrs
of pouring the gel.
Also if the gels are poured and stored in the fridge then the urea
precipitates of of the gel.
10. We do 2-3 day storage of gels by adding a moist paper towel under the
plastic wrap and then seal the plastic wrap with that infamous tape we're not
using to seal the plates with any more and still have several rolls of left.
We then have been storing them in the cold room or at room temp in a drawer
after covering with Al-foil to prevent light exposure, and they work fine
after 2-3 days.
11. Moister papertowel on both ends and bag the whole gel with clamps on in
dark plastic bag - Ok up to five days if you change the moister papertowel
every other day.
12. Removing the gel from the frame and wrapping the ends in saranwrap
('clingfilm') should keep the ends from drying out too much.
Certainly on the 373 I frequently use pretty ancient (2-3 days)
gels which
have been wrapped in this manner with no drying out to the extent that it
renders them unusable. The wells are a bit more fragile and any acrylamide
on the outside of the plates is that much more crusty to remove but
otherwise for the resolution required for genotyping it is fine. These
gels sit in the lab at room temperature in a box we had made to store both
prepared gels and together-plates which are ready for pouring into.
In my experience the 373 genotyping is pretty robust in terms of
what one
can get away with as far as gel quality goes.
