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 
methyl mercury.

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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