Hoefer HE33 User Manual download pdf (Page 20)

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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

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.

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Hoefer HE33 User Manual Page 20

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