Rna mobility, Running buffers for dna in agarose gels – Hoefer HE33 User Manual
Page 20

•
p12
Important! Do not adjust the
pH of these buffers once
they are prepared according
to the recipe!
RNA mobility
RNA can also be separated on the basis of size.
To avoid anomalies due to secondary structure,
RNA is denatured either before or during electro-
phoresis. For example, RNA fragments previously
denatured with glyoxal and dimethylsulfoxide can
be separated on neutral agarose gels, or RNA can
be fractionated on agarose gels containing meth-
ylmercuric hydroxide or formaldehyde.
RNA samples usually require longer runs or
buffers that are easily depleted, and so require
circulation. The Hoefer SUB20C and SUB25C
horizontal units are recommended for this appli-
cation rather than the HE33.
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.
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)
0.89 M
108.0 g
Boric acid (FW 61.8)
0.89 M
55.0 g
EDTA solution
(0.5 M, pH 8.0, soln. 3)
0.02 M
40.0 ml
Deionized H
2
O
to 1000.0 ml
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.
—or—
1X, to yield 89 mM Tris base, 89 mM boric acid, and
2 mM EDTA.