Purification of poly(A)+ RNA from total RNA

0. (Optional)
Before using Oligotex-dt30 recommend Oligotex-dt30 washed to remove finer particles of latex.To wash the latex, transferring the appropriate amount of Oligotex-dt30 (300 ul Oligotex suspension of 1 mg of total RNA) in a microcentrifuge tube. Spin for 3 minutes at 12,000 rpm. / Min Discard supernatant. Gently suspend the latex in the same volume of 10 mm Tris-HCl (pH 7.5), 1 millimeters, EDTA, 0.1% SDS. Spin again. Gently suspend the latex in the same buffer.

1. Add 1 mg of total RNA dissolved in RNase free water to 300 ul Oligotex-dt30 suspension.

2. Incubate for 3 minutes at a temperature of 65 C. Chill on ice.

3. Add 0.2 volume of 5M NaCl. Incubate for 10 minutes at 37 ° C.

4. Centrifuge for 3 minutes at 15,000 rpm. / Min Discard supernatant.

5. Retention of pellets in 1 ml washing buffer (10 millimeters Tris-HCl pH7.5, 1MM EDTA, 0.5M NaCl, 0.1% SDS).

6. Centrifuge for 3 minutes at 15,000 rpm. / Min Discard supernatant.

7. Retention of pellets in 300 ul water-free RNase, containing 0.1% sustainability.

8. Incuabte for 5 minutes at 65 C. Chill on ice.

9. Centrifuge for 3 minutes at 15,000 rpm. / Min Transfer supernatant into new tube microcentrifuge.

10. Make extranction phenol chloroform extraction and ethanol precipitation by standard procedure. The precipitate is washed with 75% ethanol. Dissolved in poly (A) + RNA in 10 ul RNase free water