Polyacrylamide gel fixation for DNA
buzardg at MAIL.NCIFCRF.GOV
Sun Mar 10 12:24:48 EST 1996
At 05:03 PM 3/8/96 -0700, you wrote:
>I have used before silver stain of DNA in PAGE (6% T). The basic protocol
>(from Bassam and Caetano-Annoles) calls for fixation in 10% acetic acid.
>Is this, therefore unnecessary for larger (>100 nt) ds DNA fragments? If
>so, that would save me a little bit of time. Are you also saying that
>the stained gel will have stable bands after staining, while still hydrated?
>In truth, I generally dry the gel shortly after staining anyways. :)
>So, after run I would do a short series of washes in water, then add silver
>nitrate, etc. How about it? Daniel Kim
Pardon the attempted humor in my original posted reply to your
question. We seem to spend some time in our work removing the 'black magic'
that accumulates around any 'kit' protocol. These methods are often written
for worst case scenarios and use by rank amateurs.
If you intend to recover nucleic acids from your gel, it should be
obvious to you that fixing it in place is the next worst thing you could do,
the worst thing being damaging it by acidic depurination, as you have
already noted. The majority of silver stains we have tried work better by
not acid/methanol fixing. The time savings is also obvious. NOVEX
SilverXPRESS has modified their protocol now to reflect this. We studied the
problem of citric vs acetic acid for reamplifiability, which would apply to
cycle sequencing also, in a paper by Calvert et al., 1995 in BioTechniques
18(5):782-786. We now no longer use a stop solution, because for the NOVEX
chemistry we were getting back-fading of the bands.
If you can afford it, SYBR Green II from Molecular Probes is a
fantastic substitute for silver staining in most applications, and takes
only 10-20 min of hands-off staining. Calvert has devised a way to stain a
sequencing sized gel with minimal fluid while it is attached to one plate.
Hope this is helpful,
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