mRNA preparation

(Oligotex mRNA Midi Kit, Qiagen)

1. Preheat Oligotex suspension to 37°C. Preheat Elution Buffer to 80°C.

Warm 2X Binding Buffer (65°C) to redissolve.

2. Mix total RNA (1mg in 500µl) with 500µl of 2X Binding Buffer

3. Add 55µl of Oligotex suspension.

4. Incubate for 3 min at 65°C.

5. Incubate for 10 min at room temperature.

6. Centrifuge for 2 min at maximum speed, remove SN.

7. Resuspend in 600µl of Wash Buffer.

8. Centrifuge for 1 min at maximum speed, remove SN.

9. Resuspend in 600µl of Wash Buffer. Transfer to spin column in Qiagen microcentrifuge tube (avoid taking any solid precipitate).

10. Centrifuge for 1 min at maximum speed, discard flow-through. Centrifuge again for 30 sec to eliminate resting buffer.

11. Transfer spin column to a new Qiagen microcentrifuge tube. Preheat tube for 30 sec at 80°C then add 33µl of preheated Elution Buffer. Resuspend Oligotex suspension by pipetting up and down.

12. Centrifuge for 1 min at maximum speed, transfer flow-through to a new Eppendorf tube.

13. Repeat elution twice (Final eluate is 100µl).

14. Precipitate mRNA by adding 10µl of 3M sodium acetate and 220µl of EtOH. Spin 15 min at maximum speed at room temperature. Remove SN.

15. Dry pellet in speed-vac for 7 min.

16. Resuspend in 12µl H2O. Take 2µl for OD260/280 readings (130µl Quartz cuvette).

17. Store mRNA at -20°C.