| 查看: 219 | 回复: 1 | |||
| 当前主题已经存档。 | |||
[交流]
【共享】【转载】RNA EXTRACTION METHOD FOR COTTON
|
|||
|
RNA EXTRACTION METHOD FOR COTTON Based on: Wan, C.-Y. and Wilkins, T.A. 1994. Anal. Bioch. 223:7-12. Collect young expanding leaves (or other tissue) and freeze in liquid N2 and store at -80°C. Pulverize tissue to a fine powder in a pre-cooled mortar and transfer to a glass homogenizer. Heat RNA Extraction buffer to 80°C and add to frozen ground tissue at a ratio of 5:1 (ml buffer:g tissue) and homogenize for 2 minutes. Transfer homogenate to a 50 ml Oak Ridge tube containing 0.5 mg proteinase K per ml of extraction buffer and incubate with mild agitation on a rotary shaker at 100 rpm for 1.5 hr at 42°C. Add KCl to 160 mM and chill on ice for 1 hour. Centrifuge for 20 min at 12,000g. Filter supernatant through Miracloth and precipitate the RNA overnight in 2M LiCl at 4°C. Centrifuge for 20 min at 12,000g to collect RNA pellet. Wash pellet 2-3 times with 5 ml cold 2 M LiCl until supernatant is relatively colorless. Resuspend RNA pellet in 2 ml 10 mM Tris-HCl (pH 7.5) and clarify by centrifugation for 10 min. Add Potassium Acetate (KAc, pH 5.5) to 200 mM in RNA suspension and incubate for 15 min on ice. Remove salt-insoluble material by centrifugation. Precipitate RNA overnight by adding 2.5 volumes cold 100% ethanol and incubating at -20°C. Pellet RNA, wash with 70% ethanol, dry briefly under vacuum, and suspend pellet in DEPC-treated deionized water of TE buffer. Determine yield by observing absorbance spectra between 220 and 320 nm. Yield should be about 800-1200 µg/g (RNA/fresh tissue). RNA Extraction Buffer: 200 mM sodium borate decahydrate (Borax) pH 9.0 30 mM ethylene glycol bis(ß-aminoethyl ether)-N,N´-tetraacetic acid (EGTA) 10 mM dithiothreitol (DDT) 2% polyvinylpyrrolidone, Mr 4000 (w/v) (PVP) 1% sodium dodecyl sulfate (w/v) (SDS) 1% sodium deoxycholate (w/v) 0.5% Nonidet NP-40 (v/v) (NP-40) [search]RNA EXTRACTION METHOD FOR COTTON[/search] [ Last edited by caucdenis on 2007-5-15 at 08:25 ] |
回复此楼» 猜你喜欢
越来越担心硕士毕业后没有竞争力了
已经有14人回复
-
论文的挂名怎么评判贡献？基金申请中，排名第一的挂名文章
已经有3人回复
-
申博自荐 2篇一区TOP+1篇EI+2公开发明专利 2027博士申请
已经有3人回复
-
闲聊
已经有6人回复
-
申博求助
已经有4人回复
2楼2007-05-15 12:43:25