palomb at beagle.Colorado.EDU
Sat Nov 5 18:03:56 EST 1994
bawagan at umdnj.edu (Hinayana Bawagan) writes:
> One of two methods I will use for mutagenizing my plasmid DNA is by
>hydroxylamine. I have no experience with this method but it has been
>successful for isolating temperature sensitive mutants of yeast.
> The protocol that I have states that the mutagenizing solution should be
>prepared FRESH. I would like to ask if this is a strict caveat and what is
>the reason for it. The protocol that I have appears straightforward and easy.
>To those who have experience with the method, did you have problems, tricks,
>precautions that you would like to share with me?
I've used HA and always made it up fresh. In the review article, "The
Mechanism of the Mutagenic Action of Hydroxylamines" by E. I. Budowsky
(Prog. Nucleic Acid Res. Mol. Biol. 16:125-188;1976) it is claimed that,
"Hydroxylamine, at low concentrations, readily undergoes oxidative con-
versions, the products of which intensively inactivate genetic material
in a nonspecific fashion." If I recall correctly, this includes loss of
bases and backbone cleavage. Freese and Freese (Biochemistry 4:2419;1965)
say that HA reacts with oxygen to produce nitroxyl (HNO), hydrogen peroxide
and several intermediate radicals.
I've also used methoxylamine and believe it could be stored for a while
without problems (but I'd have to do some serious double-checking before
I made any real assurances). Good luck with your mutagenesis.
>>Ehh... I could be wrong, ya know...<<
>>palomb at beagle.colorado.edu <<
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