Bind silane protocols for seq gels [A Procedure]
Dennis J. Templeton
djt2 at po.CWRU.Edu
Thu Feb 27 12:57:43 EST 1992
In a previous article, 37_527 at uwovax.uwo.ca (Gregory A. Denomme) says:
> I would like to obtain receipes for coating the outer plates for sequencing
>gels using BDH's Silane A-174 as the 'bind Silane'. I am having some trouble
>at the point of separating the plates and maybe a new protocol would help.
> Thanks in advance
>Gregory Denomme email: gdenomme at uwovax.uwo.ca
>University Hospital - BRE12
>PO Box 5339
>London, Ontario, CANADA N6A 5A5
>Tel: (519)663-3551 FAX:(519)663-3743
The other responses to this seem to have missed the point.
This is a great method for drying sequencing gells, attached to the glass,
and has been virtually foolproof for us, if you avoid the mistake of
getting the bind-silane on both plates.
I got the protocol I use from Lynn Corcoran, Now at Melbourne's WEHI, and
have probably modified it some.
We buy Sigma's M-6514 (Methacryloxypropyltrimethoxysilane) which is much
cheaper than Pharmacias A-174 and is the same thing, I think
Make up fresh: 10 ml 95% EtOH
100 ul Acetic Acid
15 ul M-6514
Wipe onto a clean, untreated glass plate with a kimwipe and gloves in a
hood, let air dry.
in the sink, rinse the entire surface of the plate 5 times with an alcohol
squirt bottle (to remove the unbound M6514, preventing it from vaporizing
onto the other plate) then let dry.
Assemble the plates just before pouring; we are careful not to allow the
plates to press against each other too. You can treat the other plate with
Rainex or siliclad if you want, but it's not crucial.
After electrophoresis, separate the plates, and your gel will be firmly
stuck to the treated plate. If the M6514 has gotten onto the other plate
it will be stuck to BOTH plates, I think this is what your problem was. Fix
the gel, plate and all as usual to remove the bulk of the urea. Rinse very
briefly with water to eliminate some of the acetic acid, then prop up the
gel overnight, or blow air on it with a fan to dry it on the glass. It
works great, dries flat, and won't be lost in the fixing process.
We buy cheap window glass and treat them as disposable plates. We have
recovered the plates when the work-study student is bored by hydrating the
gel for a few minutes, then scraping the gel onto a towel with a putty
knife, (radiowaste) and soaking the plates in 0.5 M KOH for 30 minutes.
The only problem we've had is with exposure; the glass plates don't fit
well in our metal exposure holders, but the paper ones work well.
This is definitely superior to drying the gels on paper, in my opinion.
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