PCR-SSCP loading mixture/denaturant??
Gregory S. Buzard 'Buz'
buzardg at MAIL.NCIFCRF.GOV
Fri Aug 16 13:28:19 EST 1996
Disclaimer: I am a co-author on the Hongyo et al. paper,
therefore I have dull axes to grind.
The term mercury seems to strike an unnatural fear into the
same scientists that are using ethidium bromide and
acrylamide on a daily basis. Methylmercury hydroxide is as
safe or unsafe as any chemical that you use in molecular
biology. Don't drink or sniff the formaldehyde, or the
Cost-wise, the 10 ml bottle of 1 molar MMH you get for 80$
will last even heavy users like ourselves several years.
That $80 pays for itself with just one succesful SSCP gel
that would have been a failure otherwise.
There are several alternatives that work to a lesser, but
to many, an acceptible level. Just heat denaturing in water
works amazingly well for many PCR fragments. Formamide,
NaOH, urea, and various combinations thereof, are working
reasonbly well in most labs. The most critical factor in
using these MMH alternatives is to keep the product
concentration down to prevent the product:product and
product:primer annealing rates low.
MMH is still the best, most consistant denaturant. We have
had our chemical safety people do mercury vapor testing
during our handling of stocks and the electrophoresis, and
analysis steps. Only when we open the 10 ml stock vial to
make 10 ul aliquots do we have a detectable vapor.
The U.S. government denies all responsibility for these
opinions, which fortunately are still mine.
Alex Parker wrote:
Howdy. I'm trying to get set up to do "cold SSCP" a la
Hongyo et al(NAR 21:3637-3642), and am wondering if there
is an equally good alternative to their use of ethylmercury
hydroxide as a chemical denaturant in the gel loading
buffer. They state that NaOH is inferior in performance in
their hands; anybody else's experiences would be very much
appreciated, as I'd rather not deal with this expensive and
doubtless toxic compound if it can be avoided.
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