Fixing sequencing gels
Chia Jin Ngee
Hi to all!
In my lab, like almost any molecular biology lab, we do DNA sequencing
here and there. The wierd and sometimes most fustrating part apart from
pouring gels is fixing them. I have used the standard 10% AcOH/10% MeOH
solution to fix them. Many in the institute I work in use 20% EtOH. What
are the advantages and disadvantages. If you Netters out there use any
other solutions other than the above please give me your ideas.
Back to fixing gels. Gels seem to have the ability to float off the glass
plate while fixing, even w/o intervention (eg shaking and disturbances).
Usually this results in what I call reconstructive gel surgery prior to
drying.What is the main problem here? Dirty glass plates or my lab has a
zero-G spot? I have experienced gels warping when fixing too. When I mean
warping I mean shrinks and swellings.
If you have other problems and solutions to fixing gels please send them
to me. I would be happy to compile your ideas/solutions to the above
problems or even the problems you encounter.
--
Jin Ngee, Chia Chemical Carcinogenesis Laboratory
(Genie, the OligoMan) Institute of Molecular and Cell Biology
mcbl...@leonis.nus.sg National University of Singapore
Tel: (065)772-3797 Kent Ridge Crescent
Fax: (065)779-1117 Singapore 0511
"I'm Mr Chia not Ms Chia and I know Genie is a girl's name"-Jin Ngee, Chia
Martin Leach
We have tried not fixing sequencing gels and it works fine.
Just dry the gel after running and expose.
--
..... Martin Leach Email:le...@mbcrr.harvard.edu
_|____ Dept. of Pharmacology Phone: (617) 638-5323
/ o / Boston Univ. School of Med. Fax: (617) 638-4329
_/ |-/__==/ 80 E. Concord St. (L603)
(BULLDOZER) \_ Boston MA 02118 "Not the old underpants on your
USA head.....WIBBLE" -BLACKADDER
the End
I agree. I do not fix sequencing nor RNA gels at all. If they are relatively
thick and loaded with urea, I soak them in water for about 10 minutes prior
to drying (to make drying easier). This will save your vacume appartaus
loads of wear by reducing its exposure to acetic acid.
Jim
J. Graham
PS. I think the fixing notion made it into Ed's laboratory folklore collection.
txp...@uabcvsr.cvsr.uab.edu
> Hi to all!
>
> In my lab, like almost any molecular biology lab, we do DNA sequencing
> here and there. The wierd and sometimes most fustrating part apart from
> pouring gels is fixing them. I have used the standard 10% AcOH/10% MeOH
> solution to fix them. Many in the institute I work in use 20% EtOH. What
> are the advantages and disadvantages. If you Netters out there use any
> other solutions other than the above please give me your ideas.
>
> Back to fixing gels. Gels seem to have the ability to float off the glass
> plate while fixing, even w/o intervention (eg shaking and disturbances).
> Usually this results in what I call reconstructive gel surgery prior to
> drying.What is the main problem here? Dirty glass plates or my lab has a
> zero-G spot? I have experienced gels warping when fixing too. When I mean
> warping I mean shrinks and swellings.
>
> If you have other problems and solutions to fixing gels please send them
> to me. I would be happy to compile your ideas/solutions to the above
> problems or even the problems you encounter.
>
We never fix our sequencing gels whether we are using 35S or 32P. We
just dry them directly onto Whatman #1 paper and expose them.
> > "I'm Mr Chia not Ms Chia and I know Genie is a girl's name"-Jin Ngee, Chia
>
James F. George, Ph.D.
University of Alabama at Birmingham
205-934-4261 voice
Roger F. Anderson
>Jim
>J. Graham
Sorry to say I found that it is not folk lore but it really helps. To solve
the problem of gels floating away try soaking a piece of blotting paper
in the 10% Meth 10% acetic acid solution and laying that on the gel before
you lift it. It works great. Be sure to soak the paper firtst, then lay it on.
I found this technique in Biotechniques a year or so ago. I don't recall
who wrote it but I for one am greatful. My gels dry faster and my band
resolution is a bit better. Also helps to use Long Ranger gel mix. Great
stuff indeed.
Just my $0.02.
Roger Anderson
George W Chacko
>> here and there. The wierd and sometimes most fustrating part apart from
>> pouring gels is fixing them. I have used the standard 10% AcOH/10% MeOH
>> solution to fix them. Many in the institute I work in use 20% EtOH. What
>> are the advantages and disadvantages. If you Netters out there use any
>> other solutions other than the above please give me your ideas.
I learnt this technique from a colleague who didn't have a reference
for it but there might be one. Take the siliconised plate off and
place the other plate with gel (gel on top) flat on the work surface.
Pour about a 100 ml of 10% acetic acid 10% methanol (this for our
plates, volume might vary) on to the gel. Wait ten minutes and tilt
the plate to let the fixative run off. The advantage of this method is
that you add enough juice to leach away a significant proportion of
the urea but not enough to float the gel off. Pull the gel off with
Whatman 3mm paper and dry it. There is a significant improvement in
the quality of the autorad and the time taken to dry it is also
lessened.
GC
the End
As it is virtually impossible to directly compare the same gel fixed and
unfixed, I was wondering why you felt it makes a difference ? I have
fixed some and not fixed others, and see no real differences. A collegue
here showed my two similar gels, one fixed and one unfixed, and there was
a barely perceptible softening of the bands on the unfixed RNA gel. I
don't think it would in any way affect the data. Because of the extra
time and wear on apparatus, I no longer fix.
Jim
J. Graham
John Nash
I don't fix, I just soak in water for 5 - 7 min. If I soak for any
longer the bands DO become fuzzy.
A trick to stop your gel lifting off the plates is NOT to add the gel
to a tray full of liquid, but to add the liquid to the gel in the
tray, i.e. place the gel (on its plate) in a photographic or other
wide bottomed tray, gently pour the liquid into the tray, and keep
pouring very slowly until the gel is covered. I've never lifted a gel
off the plate this way. If a corner does lift, you can blow it back
with a lungful of air.
--
John Nash (na...@nrcbsa.bio.nrc.ca)
Institute for Biological Sciences, National Research Council of Canada,
*** Disclaimer: All opinions are mine, not NRC's! ***
Chris Williams
.
I have never fixed my sequencing/genotyping gels. I usauuly don't even
dry them down. Typically I use 2 pieces of Whatman 3mm paper and wrap in
saran wrap and expose.
Vivian Miao
Ngee) wrote:
>
> If you have other problems and solutions to fixing gels please send them
> to me. I would be happy to compile your ideas/solutions to the above
> problems or even the problems you encounter.
>
> --
Oboy, a vote! I vote for not fixing gels before you dry them. My
experience is similar to those who claim that unfixed gels work fine. We
reserve the fixing step for gels that do need reconstructive surgery (e.g.
gel got torn when removing the backing plate). Then fixing makes it a lot
easier to reassemble the gel pieces in place.
Martin Kennedy
> If you have other problems and solutions to fixing gels please send them
> to me. I would be happy to compile your ideas/solutions to the above
> problems or even the problems you encounter.
>
> --
> Jin Ngee, Chia Chemical Carcinogenesis Laboratory
We never bother fixing sequencing gels; solves the problem well! Just transfer
to 3M paper and dry, works fine.
Cheers,
Martin
NNNN NN Martin A Kennedy (E-mail = mken...@chmeds.ac.nz) ZZZZZZZ
NN NN NN Cytogenetic and Molecular Oncology Unit ZZZ
NN NN NN Christchurch School of Medicine ZZZ
NN NNNN Christchurch, New Zealand ZZZZZZZ
Phone (64-3)364-0880 Fax (64-3)364-0750
daj
In our experience, we get warping when part of the gel comes off and
part stays on the plate. If just a corner comes off, we tend to leave the
rest on the plate but if it looks like a significant portion of the gel is
lifting, we encourage the whole thing to come off with gentle rocking so
it can strech evenly (and then pray it doesn't fall off when we lift the
plates out of the wash solution). We use an aspirator attached to the tap
to suck off most of the wash solution (to below the top of the plate)
before attempting to lift the plate. Some small pieces of glass (or
heavy coins) placed on the gel can help keep it in place when lifting the
plate. Similarly, if you get distortion ridges in the gel once it has been
lifted onto filter paper it is sometimes possible to flood the area with
wash solution and (gently) press the ridges down (you need to use lots of
wash solution as you are effectively trying to float the gel away from the
paper and allow it to shrink back down to size).
Obviously, this is only really safe with sulphur gels. I wouldn't like to
play around with phosphorus gels in this manner but I guess that most
phosphorus users wouldn't bother washing their gels anyway.
To try and prevent lifting in the first place, let the gel/plates
assembly cool for about 15 minutes before trying to separate them and make
sure that everyone in the lab uses silane on only one of the plates (and it
is the same plate for everyone - trying to push a gel back onto a
silanised plate is no joke).
We prewet the filter paper in the wash solution to allow it to stretch
before placing it on the gel to minimise streching artefacts (same
principle as hanging wallpaper). if you do this, you will need to put
tissues on the top surface of the plate/gel/paper assembly to suck out
excess fluid or the gel won't stick to the paper.
We use 10%methanol /10% glacial acetic, lower concentrations also tend to
encourage gel expansion.
Hope this helps - when all else fails, go away and have a cup of coffee and
try again later when you have calmed down. (the probability of loosing the
gel being directly proportional to the importance of the data on it!).
It won't hurt to leave the gel in wash for longer than the usual time
Cheers,
DAJ
David A. Johnston
Dept of Zoology, The Natural History Museum, Cromwell Road,
South Kensington, London SW7 5DB.
(tel 071 9389297, fax 071 9388754, email d...@nhm.ic.ac.uk)
Jim Owens
mken...@chmeds.ac.nz writes:
>We never bother fixing sequencing gels; solves the problem well! Just
transfer
>to 3M paper and dry, works fine.
No one in our lab has fixed a sequencing gel since 1986. We just pry the
glass plates apart, transfer to Whatman 3MM paper, and dry.
Recently, when all the gel dryers were in use, we found that gels dry
faster if laid on top of the gel dryer. The problem is to keep careless
people from putting something down on the gel while it is on the dryer.
Good luck,
Jim Owens
Jim Owens
transfer
>to 3M paper and dry, works fine.
Jim Owens
transfer
>to 3M paper and dry, works fine.
Hong Dang
It may stick to the film, use some baby powder.
Hong
Andre Hamel
Ditto! Over past 7 yrs of LOTS of sequencing, I've NEVER detected
significant diff. between fixed vs. unfixed vs dried vs undried. There is
SLIGHT difference between dried & undried if film exposed at rm temp, but
not is at -70C.
I simply lift off gel with Whatman 3MM (I prefer 21cm x 100m rolls .. < $$ :-)
Then cover with plastic wrap, expose film (-70C vs rm temp DOES make SLIGHT
difference).
best regards,
********************
Andre Hamel email: ha...@ccu.umanitoba.ca
Manitoba Veterinary Services lab tel.: (204) 945-7630
545 University Crescent, FAX:(204) 945-8062
Winnipeg, Manitoba,
CANADA R3T 5S6 ********************
Dave Harry
Just to add another comment to the general sentiment here.... Neither do
I have the patience to deal with fixing sequencing gels. But sometimes
the dried gels (ie 35S) are still sticky. If so, I sprinkle a little baby
powder (talc, haven't tried corn starch) on the gel and spread it around
with a gloved hand (just a dab, not too much). I devised this in the
Midwest, where humidy was a problem in the summer (and my daughter kept
getting diaper rash!! :-) ... ). Sometime later, I found someone else
had also reported this on the Net. I don't know how widely used this is,
but it seems to work OK and doesn't seem to cause problems with developer.
Dave Harry
Bob DeLisio
"j...@helix.nih.gov" "Jim Owens" writes:
I have air dried many sequencing gels by hanging them, like clothes, in a hood.
Works great and people usually don't mistake them for drink coasters :)
Bob
Bob DeLisio
=====================================================
_/ _/ Robert DeLisio
_/ _/ Roche Institute of Molecular Biology
_/ Roche Research Center
_/ _/ Nutley, New Jersey 07110-1199
_/ _/
_/ _/ Voice: (201) 235-3728
_/ Fax: (201) 235-2318
_/ _/ Internet: deli...@rnisd0.dnet.roche.com
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=====================================================
srg...@grv.grace.cri.nz
>unfixed, I was wondering why you felt it makes a difference ? I have
>J. Graham
I agree that result-wise it impossible to tell the difference between unfixed
and fixed gels PROVIDED the unfixed gel doesn't stick to the Xray film. I
prefer to fix sequencing gels since I find unfixed ones take longer to dry and
always seem a bit tacky and prone to sticking to the X-ray film.
Sequencing gels are tougher than you may think. If they come of the backing
plate in washing you can sort them out by adding more fixative and taking a
lot of care. Another pair of hands helps. I've got some potentail disasters to
work ok.
PS Hi to other Kiwi net readers, esp Martin K, Tania S and Andrew Dog-breath
Fellowes
Brian Scrimshaw
Wellingtn
New Zealand
Ludwig Lab-UCSC
I gave up fixing gels years ago. I dry them directly onto
3mm paper and autorad them as is. The urea probably fuzzes the bands
a little, but the gels are =very= readable....
--b0b kuhn
>
> Hi to all!
>
> In my lab, like almost any molecular biology lab, we do DNA sequencing
> here and there. The wierd and sometimes most fustrating part apart from
> pouring gels is fixing them. I have used the standard 10% AcOH/10% MeOH
> solution to fix them. Many in the institute I work in use 20% EtOH. What
> are the advantages and disadvantages. If you Netters out there use any
> other solutions other than the above please give me your ideas.
> warping I mean shrinks and swellings.
Stuff deleted
> If you have other problems and solutions to fixing gels please send them
> to me. I would be happy to compile your ideas/solutions to the above
> problems or even the problems you encounter.
>
> --
> Jin Ngee, Chia Chemical Carcinogenesis Laboratory
> (Genie, the OligoMan) Institute of Molecular and Cell Biology
> mcbl...@leonis.nus.sg National University of Singapore
> Tel: (065)772-3797 Kent Ridge Crescent
> Fax: (065)779-1117 Singapore 0511
> "I'm Mr Chia not Ms Chia and I know Genie is a girl's name"-Jin Ngee, Chia
------------------------------------------------
Robert Kuhn, PhD
Sinsheimer Laboratories
University of California
Santa Cruz, CA 95064 USA
email: rk...@orchid.ucsc.edu
================================================
Ludwig Lab-UCSC
> >unfixed, I was wondering why you felt it makes a difference ? I have
> >Jim
> >J. Graham
>
> I agree that result-wise it impossible to tell the difference between unfixed
> and fixed gels PROVIDED the unfixed gel doesn't stick to the Xray film. I
> prefer to fix sequencing gels since I find unfixed ones take longer to dry and
> always seem a bit tacky and prone to sticking to the X-ray film.
I always leave the Saran Wrap/cling film on the gel in the cassette.
The 35S signal is reduced somewhat, but it never sticks to the film.
--b0b kuhn