MRNA Prep

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Materials Required for Poly(A) mRNA Isolation With Sera-Mag Oligo(dT) Beads

• 48 well plate or RNase-free, non-sticky 1.5 mL tubes

• Sera-Mag Oligo(dT)14 beads, Seradyn #2815-2103-010150 (Now from Thermo-Forma; 20 µL per sample)

• Bead Binding Buffer (20mM Tris-HCl pH 7.5, 1.0M LiCl and 2mM EDTA; user supplied, 330 µL per sample)

• Bead Washing Buffer (10mM Tris-HCl PH 7.5, 0.15M LiCl, 1mM EDTA; user supplied, 700 µL per sample)

• 10 mM Tris-HCl, pH 8 (user supplied, 40 µL per sample)

• magnetic stand for capturing beads (either 1.5 mL or multiwell plate format)

Method for Poly(A) mRNA Isolation With Sera-Mag Oligo(dT) Beads

1. Set pre-heat two heat blocks, one at 65°C and the other at 80°C (or use a PCR machine).

2. Dilute 1-10 µg of total RNA with nuclease-free H2O to a final volume of 50µL in a 1.5 mL RNase-free microfuge tube or deep-well 48 well plate.

3. Heat samples at 65°C for 5 minutes to disrupt the secondary structures; snap-cool and store on ice.

4. Meanwhile, mix the Sera-Mag Oligo(dT)14 beads well, and aliquot 20µL of beads into each 1.5 mL RNase-free microfuge tube or deep-well 48 well plate. Place tube on magnetic stand and remove the supernatant.

5. Add 100µL of Bead Binding buffer (B2 buffer: 20mM Tris-HCl pH 7.5, 1.0M LiCl and 2mM EDTA) to each tube containing Sera-Mag beads.

6. Vortex briefly, then place tube on magnetic stand.

7. Repeat the steps 5 and 6 for a total of two B2 Buffer washes.

8. Remove the tube from the magnetic stand and resuspend the beads in 50µL of B2 Buffer.

9. Add the 50µL of total RNA sample from step 3 and mix well.

10. Mix and rotate the tube from step 9 at RT for 5 minutes.

11. Place the tube from step 10 on the magnetic stand. Remove and discard the supernatant. Do not disturb beads.

12. Remove the tube from the magnetic stand and add 200µL of Bead Washing buffer (BW buffer: 10mM Tris-HCl PH 7.5, 0.15M LiCl, 1mM EDTA). Gently vortex the tube to mix.

13. Place the tube on the magnetic stand, then remove and discard the supernatant. Do not disturb the beads.

14. Repeat steps 12 and 13 for a total of two BW Buffer washes.

15. Add 50µL of 10mM Tris-HCl to the tubes from step 14. Gently vortex to mix thoroughly.

16. Aliquot 50µL of B2 Buffer to a new 1.5 mL RNase-free microfuge tube or deep-well 48 well plate.

17. Heat the sample from step 15 on a preheated PCR machine at 80°C for 2 minutes to elute mRNA from the beads.

18. Immediately put the beads on the magnet stand, and transfer the supernatant (mRNA) to the tube/plate from step 16 (the one containing B2 Buffer).

19. Heat the mRNA sample from step 18 at 65°C for 5 minutes to disrupt the secondary structures, and place the tube on ice.

20. Wash the beads from step 18 by resuspending them in 200 µL of BW Buffer. Gently vortex to mix.

21. Magnetize, then remove and discard the supernatant from the tube. Do not disturb the beads.

22. Repeat steps 20 and 21 for a total of two BW Buffer washes.

23. Add 100µL of mRNA sample from step 19 to the beads.

24. Mix and rotate the tube at RT for 5 minutes, then magnetize.

25. Remove and discard the supernatant from the tube. Do not disturb the beads.

26. Wash beads with 200µL BW Buffer. Gently vortex to mix beads thoroughly.

27. Magnetize, then remove and discard the supernatant from the tube.

28. Repeat steps 26 and 27 once for a total of two BW Buffer washes.

29. Add 20 µL of 10mM Tris-HCl to the tube from step 28.

30. Heat the tube from step 29 on a pre-heated PCR machine at 80°C for 2 minutes to elute mRNA from the beads.

31. Immediately place the tube on the magnetic stand and transfer all of the supernatant (mRNA) to a new thin wall 0.2 mL RNase-free microfuge tube or deep-well 48 well plate.well.


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