Conifer RNA prep

Materials Required for Total RNA Extraction from Douglas-fir Needles
''Modified Urea/LiCl buffer* (8 M Urea, 3 M LiCl, 1% polyvinylpyrrolidone K-60, 5 mM DTT (add just before use, 1M stock). Modified from Tai et al., 2004, Plant Molecular Biology Reporter 22:93a-93e.

Extraction buffer, 50 ml: Add in this order…

• 20 mL 7.5 M LiCl solution

• 24 grams Urea

• 5 mL 10% PVP K-60 stock solution

'' add water up to 49.5 mL, mix well (heating to 50C may help)

 just before use, add:

• 0.5 mL of 1 M Dithiothreitol

7.5 M LiCl:  • Ambion RNA Precipitation Solution

Tubes:  •	Fast-Prep grinding tubes (1 per sample, 2 beads each) •	2 mL centrifuge tube (1 per sample) •	1.5 mL centrifuge tube (2 per sample)

Zymo supplies •	Zymo-spin IIIC Column (1), Zymo-spin IIC column (1), collection tube (1), 1.5m L tube (1), RNA Lysis Buffer, RNA Prep Buffer, RNA Wash Buffer (with EtOH added)

Reagents and Materials •	DEPC Water, 10% PVP K-60, 1 M DTT, 100% EtOH, 70% EtOH, RNA Storage Solution, Dry Ice flakes or chips (about 5 lbs), water ice.

Method for Total RNA Extraction from Douglas-fir Needles
1. Samples (100 mg-200 mg) should be placed in a Fast-Prep grinding tube containing two ceramic beads (with or without garnet – doesn’t matter)

2. Add flaked dry ice to the 24 position Fast-Prep teflon grind adaptor. Grind tissues using two pulses of 20 seconds each, angular momentum of 4. Make sure that the tissue stays frozen and that it is ground to a fine powder.

3. After grinding, add 1 mL of COLD (4°C) Urea/LiCl RNA Extraction Buffer to each tube. Shake vigorously or Fast-Prep for 10 additional seconds, until the ground dried tissue is completely resuspended.

4. Place the tubes in the centrifuge; spin at 200g x 10 min at 4°C to pellet the cellular debris.

5. Transfer 0.7 mL of supernatant to a clean 1.5 mL RNase-free tube.

6. Incubate supernatant mixture overnight on ice in the 4°C refrigerator.

 Day 2 ---

7. Centrifuge solution at 20,000g x 30 min, 4°C.

8. Discard the supernatant carefully. Rinse the pellet in 200 μl of 70% ethanol, the re-centrifuge at 20,000g x 5 minutes, room temperature.

9. Discard the supernatant carefully. Let the pellet briefly air dry 5 minutes.

10. Thoroughly resuspend the RNA pellet in 400 μl of Zymo RNA Lysis buffer, then resume the Zymo process at step 4.

 For simplicity, here is the Zymo process ---

11. Transfer all 400 μl of the RNA/RNA Lysis buffer into a Zymo-Spin IIIC column in a collection tube and centrifuge at 8000g x 30 seconds. Save the flow-through.

12. Add 0.8 volumes (320 μl) of 100% ethanol to the sample/flow-through and mix well.

13. Transfer the mixture to a Zymo-Spin IIC column in a collection tube. Centrifuge at > 12000g x 30 seconds. Discard flow through.

14. Add 400 μl of Zymo RNA Prep Buffer to the column. Centrifuge at > 12000g x 60 seconds. Discard flow through, and replace Zymo-Spin IIC column back into the same collection tube.

15. Add 800 μl Zymo RNA Wash Buffer to the column. Centrifuge at > 12000g x 30 seconds. Discard flow through, and replace Zymo-Spin IIC column back into the same collection tube. Repeat the wash with 300 μl RNA Wash Buffer.

16. Centrifuge the Zymo-Spin IIC column at > 12000g x 2 minutes in the empty collection tube to ensure complete removal of the wash buffer.

17. Carefully remove the Zymo-Spin IIC column from the collection tube and place into an RNase free tube. Add 25 μl of RNA Storage Solution (Ambion) directly to the column; let stand one minute.

18. Centrifuge the Zymo-Spin IIC column at 10000g x 30 seconds to elute RNA.

19. Estimate RNA concentration using Qubit.

20. If DNA needs to be removed, treat with DNA-free Turbo kit (Ambion).

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