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Important! Do not adjust the
pH of these buffers once they
are prepared according to the
recipe!
p14
•
RNA mobility
RNA can also be separated on the basis of size.
To avoid anomalies due to secondary struc-
ture, RNA is denatured either before or during
electrophoresis. For example, RNA fragments
previously denatured with glyoxal and dimeth-
ylsulfoxide can be separated on neutral agarose
gels, or RNA can be fractionated on agarose gels
containing methylmercuric hydroxide or formal-
dehyde.
RNA samples usually require longer runs or
buffers that are easily depleted, and so require
circulation. The Hoefer HE 100 horizontal unit
is recommended for this application rather than
the HE 33.
Running buffers for DNA in agarose gels
Recipes for the two most commonly used
running buffers for DNA electrophoresis are
listed below. The ionic strength of these buffers
is appropriate for the application; do not adjust
the pH of these buffers once they are prepared
according to the recipe!
1. 10X Tris-borate-EDTA (TBE) stock buffer
(0.89 M Tris, 0.89 M boric acid, 20 mM EDTA,
pH ∼8.2, 1000 ml)
Tris base (FW 121.1)
Boric acid (FW 61.8)
EDTA solution
(0.5 M, pH 8.0, soln. 3)
Deionized H
O
2
Stir. Do not adjust pH.
Before use dilute either to:
0.5X, to yield 45 mM Tris base, 45 mM boric acid,
and 1 mM EDTA. This dilution is often used because
current remains low, resulting in less heat.
0.89 M
108.0 g
0.89 M
55.0 g
0.02 M
40.0 ml
to 1000.0 ml
†
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