神马文学网Arabidopsis RNA extraction protocol
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发布日期:2009-7-24 热门指数:1340收藏
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.
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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