拟南芥RNA提取方法--Arabidopsis RNA extraction protocol

Arabidopsis RNA extraction protocol Based on Krapp at al (1993) Plant Journal 3:817 with modifications.Comments or Questions? Send to me at: chunming.liu@wur.nl 1-2 g fresh material, freezer-dried, ground with 0.2g sand (if necessary), and then homogenized with 10ml RNA extraction buffer (see below). Spin at 8,000rpm, 4o C, for 10 minutes Remove the supernatants to new tubes, phenol/chloroform extracted (8,000rpm at 4o C 10’). Wash the supernatant with 10 ml chloroform (8,000rpm at 4o C, 10’). Add 1/10 vol of 3M NaAc (700ul), and 2 vol of ethanol (15ml), -80o C 2hrs. Centrifuge at 8,500rpm for 30min at 4o C, and discard the supernatant. Resuspend the pellet in RNA resuspension buffer (see below), 4 o C 1hr. Centrifuge at 8,500rpm for 10min at 4o C, 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 4o C.

• 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 4o C for use.

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