Hoefer TE42 User Manual
Page 30
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Nucleic acid transfers
Nucleic acids must normally be transferred in
denatured form for most efficient binding. RNA
is normally denatured with glyoxal before sepa-
ration or separated in denaturing gels contain-
ing formaldehyde or methyl mercury. However,
double stranded DNA is usually denatured in
the gel with NaOH. The alkali must be neutral-
ized and the gel equilibrated in transfer buffer
before electrotransfer. For both DNA and RNA
gels, any SDS must also be removed to assure
efficient binding. Bittner et al. (1980) wash gels
three times, 20 minutes each, to assure complete
removal of denaturants and detergents.
See Bittner et al. for a study of the transfer effi-
ciency for DNA of different sizes. The Bittner
transfer buffer contains 25 mM sodium phos-
phate, pH 6.5. Also described is a method for
the introduction of nicks by limited nuclease
action in order to facilitate transfer of larger
DNA fragments.
Recommended DNA buffers include the Bittner
sodium phosphate buffer (see reference) and
TBE. For RNA, TAE is recommended. TBE
and TAE stock recipes are listed below. These
buffers are most often diluted to 1X, but the
concentration can range down to 0.1X. Cooling
is strongly recommended for these buffers, espe-
cially at higher concentrations.