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1. Mix 600 µl (1 mg) total RNA in nuclease-free ddH2O
with 600 µl 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 µl 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 µl 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 µl mRNA fractions.
12. Precipitate with ethanol .
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