Mullins Molecular Retrovirology Lab

  • Department of Microbiology
  • School of Medicine
  • University of Washington
University of Washington/Fred Hutch Center for AIDS Research

Preparation of Linear Acrylamide

Unlike glycogen, linear acrylamide does not inhibit polymerase activities, or restriction enzymes.

Short Protocol:

  1. Dissolve 5mg Acrylamide in 200 µl TE

  2. Add 1 µl 10%APS

  3. Add 1 µl TEMED

  4. Allow to polymerize (incubate as long as you like, can heat to 37°C to increase rate): forms a jelly mass.

  5. Add 2.5 volumes ethanol.

  6. Spin 5-6 minutes.

  7. Speed vac to dry.

  8. Resuspend 500 µl ddH20 (i.e. 10 µg/µl). Takes a while to resuspend (may leave at RT overnight). Subsequent pellets are easy to resuspend.

  9. Use 10-20 µg for a precipitation.

Linear poly-acrylamide can be used as an efficient neutral carrier for precipitating nucleic acids with ethanol (Gaillard, C and Strauss, F. Nucleic Acids Res. 18, 378). Alhough 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.

Extended Protocol:

  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 in 10 mM Tris-HCl (pH 8.0), 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.

  8. Picogram amounts of nucleic acids longer than 20 base pairs can be precipitated without loss.