reusing agarose gels
Diana Sammataro
Hello fellow RAPDs people.
I am working with honey bees, spiroplasma and mites and have found
a good protocol (when the RAPDs god/goddess are cooperating) to
extract the DNA using Longmire's bird blood protocol and dialysis
plus salt precipt.
Recently, some of my primers seem to be failing since I have just
purchases new reaction mixes, made my own MgCl and use tissue cultured
water. I also UV pipettors, tips, and tubes as well as the water
to get rid of negative control bands.
A few questions:
1.Can I reuse agarose gels, and what happens to DNA and EB in the
gels.
2. What is a positive control?
As my system is shutting down in a few minutes I will sign off
for now.
Please reply
Diana Sammataro, Dept Entom. dsam...@magnus.acs.ohio-state.edu
David Huen
dsam...@magnus.acs.ohio-state.edu (Diana Sammataro) wrote:
>
<stuff deleted>
>
> 1.Can I reuse agarose gels, and what happens to DNA and EB in the
> gels.
Yes, just melt it down (+ wee bit of water) and recast it. Both are still
there. The next time round, you will not need to stain the gel as the EB is
still there. Boiling denatures DNA and ssDNA doesn't fluoresce as much with
EB. Still, the background rises with increasing recycling (some bits of
dirt will also fluoresce...). I've done 4-5x recycling out of sheer sloth
before...
DO NOT USE RECYCLED GELS FOR SOUTHERN BLOTTING!!!!!
Paul St. Amand
(David Huen) wrote:
We simply run the DNA off of the gel (by electrophoresis) after
photographing. The gel can then be reused without melting and recasting. We
add EtBr to the buffer or add it to the wells with the sample. This is
great for those expensive 4% agarose gels. If your application doesn't have
too many bands, you don't even need to run the old DNA off the gel. Just
make sure that new bands aren't migrating over old ones. I have reused a
gel this way at least 5 times and it seems like it can take many more runs.
The only trouble is with low % agarose (0.6 to 1.5 %) gels, they tear
easily with the repeated handling.
Paul C. St. Amand Research Geneticist
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
USDA/ARS and KSU Agronomy Dept. P...@KSU.KSU.Edu
(913) 532-6168 Fax (913) 532-5692
the End
I return my gels to the electrophesis rig and run the sample through
prior to reusing the same whenever I am particularily lazy :)
Jim
J. E. Graham
Lauri Lintott
> In article <2tk8sp$a...@charm.magnus.acs.ohio-state.edu>,
> dsam...@magnus.acs.ohio-state.edu (Diana Sammataro) wrote:
> >
>
> <stuff deleted>
> >
> > 1.Can I reuse agarose gels, and what happens to DNA and EB in the
> > gels.
> Yes, just melt it down (+ wee bit of water) and recast it. Both are still
> there. The next time round, you will not need to stain the gel as the EB is
> still there. Boiling denatures DNA and ssDNA doesn't fluoresce as much with
> EB. Still, the background rises with increasing recycling (some bits of
> dirt will also fluoresce...). I've done 4-5x recycling out of sheer sloth
> before...
>
> DO NOT USE RECYCLED GELS FOR SOUTHERN BLOTTING!!!!!
>
>
vaporize and be inhaled, and since it is a mutagen/carinogen this
would not be a good thing. Does someone know better?
Lauri Lintott
Bio. Sci. U of Calgary
Cornelius Krasel
: I was always told that boiling ETBr is a bad idea as it can
: vaporize and be inhaled, and since it is a mutagen/carinogen this
: would not be a good thing. Does someone know better?
In the lab where I was working previously once somebody put some
EtBr-containing vials into the autoclav. After autoclaving, the
person who opened it got an anaphylactic shock (fortunately, she
survived without any damage). Later, it became apparent that she
was allergic against Br-containing substances, and the shock was
apparently caused by EtBr vapours.
--Cornelius.
--
/* Cornelius Krasel, Abt. Lohse, Genzentrum, D-82152 Martinsried, Germany */
/* email: kra...@alf.biochem.mpg.de fax: +49 89 8578 3795 */
/* "People are DNA's way of making more DNA." (Edward O. Wilson, 1975) */
Armadin
dsam...@magnus.acs.ohio-state.edu (Diana Sammataro) wrote:
>
> A few questions:
>
> 1.Can I reuse agarose gels, and what happens to DNA and EB in the
> gels.
>
we have recast 1% agarose gels over 40 times with the only side effect
being the gel gets brittle. after taking the picture we put the gel (150 ml
1%) in a beaker with 1% TBE and let it soak. this removes most of the dye
(orange G for us) and probably some of the EtBr. saving on agarose is
imoportant to us since we can run 120 gels a week when we are in production
mode. the background level of recycled gels increases so you have bright
gels, i add more EtBr each time. i would not use recycled gels for
preparative purposes but it is fine for analytical gels. as for EtBr
vaporization concerns, we check our microwave every now and then and have
not noticed a build up of EtBr on the inside.
--
robert lagace
lag...@camis.stanford.edu
armadin.aol.com
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