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Star Republic: Guide for Biologists

RNA purification --- polyA (I)

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1. Mix 600 l (1 mg) total RNA in nuclease-free ddH2O with 600 l 2X binding buffer (1 M NaCl, 20 mM Tris pH 7.5, 2 mM EDTA, 0.1 % SDS).

2. Add 40 mg oligo-dT cellulose in 1X binding buffer.

3. Heat 3 minutes at 70 C to denature the RNA.

4. Cool to room temperature 10 minutes to allow annealing of mRNA to resin.

5. Pellet the resin containing bound mRNA by spinning for 2 minutes in a microfuge.

6. Resuspend the resin in 600 l of wash buffer (0.2 M NaCl, 10 mM Tris pH 7.5, 1 mM EDTA, 0.05 % SDS) by vortexing vigorously.

7. Pellet the resin by centifuging in a microfuge at the maximum speed.

8. Repeat washing steps three times.

9. Elute with 250 l elution buffer (10 mM Tris pH 7.5, 1 mM EDTA, 0.05 % SDS) at 37 C for 10 minutes.

10. Repeat step 9.

11. Combine the two 250 l mRNA fractions.

12. Precipitate with ethanol

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