ArabidopsisRNAextractionprotocol

1. 1-2 g fresh material, freezer-dried, ground with 0.2g sand (if necessary), and then homogenized with 10ml RNA extraction buffer (see below).

2. Spin at 8,000rpm, 4^oC, for 10 minutes

3. Remove the supernatants to new tubes, phenol/chloroform extracted (8,000rpm at 4^oC 10’).

4. Wash the supernatant with 10 ml chloroform (8,000rpm at 4^oC, 10’).

5. Add 1/10 vol of 3M NaAc (700ul), and 2 vol of ethanol (15ml), -80^oC 2hrs.

6. Centrifuge at 8,500rpm for 30min at 4^oC, and discard the supernatant.

7. Resuspend the pellet in RNA resuspension buffer (see below), 4 ^oC 1hr.

8. Centrifuge at 8,500rpm for 10min at 4^oC, resuspend in RNase-free water (1 to 1.5ml).

Solutions:

• RNA extraction buffer:

• 4M Guanidine thiocyanate

• 20 mM EDTA

• 20 mM MES

• Adjust pH to 7.0

• Add RNase-free water to final volume 400 ml, filtrate and autoclave, store at R.T.

• Add 1.7ml (the final concentration is 50 mM) 2-mercaptoethanol to each 400ml solution and store at 4^oC.

• RNA resuspension buffer:

• 2M Lithium Chloride

• 10mM Sodium Acetate

• Adjust final volume to 250 ml and pH to 5.2

• Filtrate and autoclave, store at 4^oC for use.