1) Pre-equilibrate oligo-dT cellulose:

a. Weigh 0.2 g per mg of RNA into a sterile Corex tube

b. Add 10ml of 0.1 M NaOH, vortex, Spin in HB4 rotor, 4K, @ RT, for 5 min.

c. Decant, add 10 ml of 1X binding buffer, vortex, spin and decant.

d. Repeat step c above 5X.

e. Decant final wash, warm to 68º C.

2) Keep RNA in small volume and bring to 1X binding buffer with 2X stock.

3) Heat RNA to 68º C, for 5 min.

4) Add RNA to oligo-dT, shake on wrist action shaker for 1-2 hrs. @ RT.

5) Spin 4k, @ RT, for 5 min.

6) Carefully remove supernatant and save for poly-A- fraction.

7) Resuspend pellet in 10 ml 1X binding buffer, spin, decant super.

8) Repeat step 7 above 7-10X

9) Elute bound RNA with 1X 0.5 ml water

3X 0.5 ml elution buffer

1X 0.5 ml water

10) Pool eluted RNA, spin @ 10K to get rid of any oligo-dT that was carried over. Note that a small amount of carry over at this point may be useful as a carrier. If a large amount is present, it may be prudent to wash it with water to elute any freshly bound poly-A RNA.

11) Precipitate RNA with 0.1 Vol. 3M NaOAC, pH 5.2 (or NH4OAC) and 3 Vol. EtOH.

1M NaCl 40 ml of 5M

0.02M Tris-HCl pH 7.5 4 ml of 1M

2mM EDTA 1.6 ml of 0.25M

0.2% SDS 4 ml of 10%

Water to 200 ml

20X Resuspension/Elution Buffer:

0.5M Tris-HCl pH 7.5 50 ml of 1M

0.02M EDTA 8 ml of 0.25M

Water to 100 ml