LinearPolypolyacrylamide

Linear Polypolyacrylamide
5% acrylamide solution(withoutbis-acrylamide)in 40mM Tris-HCl,20 mM Na Acetate,1 mM EDTA,pH7.8. Add 1/100 vol.of 10% 
ammonium persulfate and 1/1000 vol. TEMED, and letpolymerize for 30min. When the solution has become viscous, 
precipitate the-polymer with 2.5 vol. ethanol, centrifuge,and redissolve the pellet in 20 vol. of water by shaking overnight.
The 0.25% linear polyacrylamide solution obtained can be stored in the refrigerator for several years.

Precipitation of DNA
to DNA in >0.1M salt,add 10-20 ug of linearpolyacrylamide and 2.5 vol. ethanol.
Leave at-70°C in a dry ice for 10min.
centrifuge for 10 min. at top speed in a microcentrifuge.
Carefully remove the supernatant, wash the pellet with ethanol and dry.
Polyacrylamide can also be used to precipitate RNA with ethanol or proteins with acetone

To produce a 5 ug/ul stock of linear polyacrylamide (from T. Fitzwater, ref 1, below):

In a 50 mL Falcon 2098 centrifuge tube, mix in the order listed (makes a lot, so may want to scale down to 10%):

0.25 g acrylamide (no bis-acrylamide) 4.25 mL of Millipure water 200 µL of 1 M Tris-HCl, pH 8.0 33 µL of 3 M sodium acetate, pH 7.5 10 µL of 0.5 M EDTA 50 µL of 10% APS (prepare 10% solution in sterile di-water) 5 µL of TEMED and mix well to begin polymerization; leave at room temperature 30 mins. Add 12.5 mls 95% ETOH to precipitate approximately 5 mins.

Drain off liquid from precipitated LPA. Remove as much liquid as possible with pipet (squash pellet).

Wash pellet with 70% ETOH, then remove as much liquid as possible with pipet (squash pellet).

Air dry about 10 mins.

Add sterile di-water to LPA, let resuspend overnight with gentle shaking. Make final volume 50 mls. Concentration of LPA is 250 mg/50 mls, or 5 mg/ml, or 5 ug/ul.

Aliquot and store at -20.

Use 3 ul in a large eppy for ethanol precipitations (15 ug LPA). Be carful to not lose pellet, as it may not stick to tube as tightly as with nucleic acid carriers.

Another web protocol:

1. See LPA recipe and notes from tfitzwater (Tim Fitwater): http://molecularbiology.forums.biotechniques.com/forums/viewtopic.php?t=999&highlight=linear+polyacrylamide

2. http://ubik.microbiol.washington.edu/protocols/arrays/linearacrylamide.htm

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

Short Protocol:

Dissolve 5mg Acrylamide in 200 µl TE

Add 1 µl 10%APS

Add 1 µl TEMED

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

Add 2.5 volumes ethanol.

Spin 5-6 minutes.

Speed vac to dry.

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.

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:

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

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

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

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

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.

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

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.

• Gaillard, C. and Strauss, F. (1990) Nucleic Acids Res. 18, 378