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Star Republic: Guide for Biologists

RNA purification --- polyA (II)

This protocol uses the Oligotex mRNA Midi Kit (Qiagen).

1. Mix cell lysate (600 l in OCL buffer for 1-5x106 cells) or 600 l (1 mg) total RNA with 600 l 2X binding buffer.

2. Add 35 l Qiagen Oligotex-dT resin.

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 by vortexing vigorously.

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

8. Resuspend the resin in 200 l of QL1 wash buffer.

9. Add 800 l of ODB buffer.

10. Pellet the resin and then resuspend in 600 l of OW1 buffer.

11. Transfer the resin to a Qiagen spin column.

10. Centrifuge at maximum speed for 30 seconds, discard flow-through.

11. Wash with 600 l of OW2, discard flow-through.

12. Repeat step 11 once.

13. Elute with 33 l 80 C elution buffer OEB.

14. Repeat step 13 twice.

15. Combine the three 33 l mRNA fractions.

16. Precipitate with ethanol if necessary.