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Colony Cracking

Preparation of linear polyacrylamide used as carrier in ethanol precipitation of nucleic acids

Linear polyacrylamide can be used as an efficient neutral carrier for precipitating nucleic acids with ethanol (Gaillard, C. and Strauss, F. Nucleic Acids Res. 18, 378). Although glycogen is often used as carrier in ethanol precipitation, it has been shown that glycogen inhibits the activity of an ssDNA-binding protein on which linear polyacrylamide has no effect.
Disadvantage using linear polyacrylamide as carrier is that the pellet of polyacrylamide does not stick tightly on the bottom of microfuge tube. Be careful not to discard pellet when you remove supernatant.

1. Prepare a 5% acrylamide solution without bis-acrylamide in 40 mM Tris-HCl (pH. 8), 20 mM sodium acetate, 1 mM EDTA.

2. Add ammonium persulfate to a final concentration of 0.1% (w/v).

3. Add 1/1000 volume of TEMED, and let polymerize for 30 min at room temperature.

4. When the solution has become viscous, precipitate the polymer with 2.5 volumes of ethanol.

5. Recover the polymer by centrifugation, and dissolve the pellet in10 mM Tris-HCl (pH8.0), 1 mM EDTA to make a final polyacrylamide concentration of 5 mg/ml.

6. The linear polyacrylamide solution can be stored in the refrigerator for several years.

7. Add 10-20 micrograms of the linear polyacrylamide into nucleic acid solution before ethanol precipitation. Picogram amounts of nucleic acids longer than 20 base pairs can be precipitated without loss.

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