Grind roots +/-, shoots +/- in ball mill (Sam's Retch Mixer Mill MM400)

• Precool the white block for the ball mill

• prepare 2 2-mL tubes per plate (label tubes on side as well as top)

  • 3 roots + iron
  • 3 roots - iron
  • 3 shoots + iron
  • 3 shoots - iron

• 2 metal beads per tube 3.2mm diameter ss beads (90g) - Cat. No. 11079132ss from www.biospec.com (Can also get from Fisher)

• cool in LN2

• Add tissue to cold tube, put back in LN2

• Put all tubes in cold block

• Shake 20 sec, check. The heat of shaking may start to defrost the tissue. Throw it back in the LN2. (Shake too long, and the cap may disintegrate!

• Return to LN2

Prepare RNA from powdered frozen tissue

Use RNeasy Plant Mini Kit (50) Qiagen 74904

1. Add 450 µL Buffer RLT. Mix vigorously. Spin down.

2. Transfer lysate to lilac Qiashredder in 2mL collection tube

3. Spin 2 min, full speed

4. Transfer flow-through (NOT PELLET) to new tube. Toss the lilac column

5. Add 225 µL 100% EtOH to lysate. Mix by pipetting up and down

6. Transfer entire sample to pink spin column in a 2 mL collection tube.

7. Discard flow-through. Keep column.

8. Add 350 µL RW1 to column. Spin 15 sec, full speed.

9. Discard flow-through. Keep column

10. Add 70µL RDD to 10 µL DNAse I. BE GENTLE. Put the 80 µL on the column. Incubate at room temp 15 min.

11. Add 350 µL RW1. Spin 15 sec. full speed.

12. Discard flow-through, reuse collection tube.

13. Add 500 µL RPE to column. spin full speed 2 min.

14. Put column in new capless collection tube. Discard old collection tube.

15. Spin 1 min, full speed

16. Add 30 µL RNAse-free water to column membrane. Spin 1 min, full speed.

17. Repeat, with 20 µL RNAse free water.

18. Transfer all 50 µL RNA to clean, labeled tube. Keep on ice

1. Clean the pedestals

2. Blank the machine

   1 µL buffer onto lower measurement pedestal

   F3 (Blank)

3. Wipe again

4. Test the blank:

   1 µL buffer

   F1 (Measure)

   if it's flat, you're good to go

5. 1 µL sample. repeat.