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- 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, 4oC, for 10 minutes
- Remove the supernatants to new tubes, phenol/chloroform extracted (8,000rpm at 4oC 10’).
- Wash the supernatant with 10 ml chloroform (8,000rpm at 4oC, 10’).
- Add 1/10 vol of 3M NaAc (700ul), and 2 vol of ethanol (15ml), -80oC 2hrs.
- Centrifuge at 8,500rpm for 30min at 4oC, and discard the supernatant.
- Resuspend the pellet in RNA resuspension buffer (see below), 4 oC 1hr.
- Centrifuge at 8,500rpm for 10min at 4oC, 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 4oC.
- 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 4oC for use.
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Pollen Development&