Synthesis of 5’ end-labeled RNA
5'末端标记RNA的合成
发布: 2012年07月20日第2卷第14期 DOI: 10.21769/BioProtoc.241 浏览次数: 13668
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参见作者原研究论文
The authors used this protocol in:
Dec 2011
Abstract
5’ end-labeled RNA molecules are useful substrates to analyse the endo- and exonucleolytic activities of various ribonucleases. Here two protocols are given to synthesize P32 labeled RNAs with a 5’ PPP or 5’ P moiety. 5’ exoribonucleases generally do not work on 5’ PPP RNA and require a 5’ P substrate. The activity of certain endoribonucleases like Escherichia coli (E. coli) RNase E or Bacillus subtilis (B. subtilis) RNase Y can be stimulated by a 5’ P moiety.
RNA Synthesis (RNA合成)
Radioactive labeling (放射性标记)
Materials and Reagents
- RQ1 DNase (Promega Corporation, catalog number: M6101 )
- Calf intestinal phosphatase (F. Hoffmann-La Roche, catalog number: 713023 )
- Phenol/Chlorophorm (1:1) (MP Biomedicals, catalog number: AQUAPH01 )/
(Carlo Erba, catalog number: 438601 )
- T7 RNA polymérase (Promega Corporation, catalog number: P207B )
- T4 Polynucleotide Kinase (Biolabs, catalog number: M0201 )
- RNasin RNase inhibitor (Promega Corporation, catalog number: N2611 )
- DTT (Promega Corporation, catalog number: P117B )
- NTPs (F. Hoffmann-La Roche, catalog number: set1277057 )
- γ32P GTP (6,000 Ci/mmole, 10 μC/μl) (PerkinElmer, catalog number: BLU504Z )
- γ32P ATP (3,000 Ci/mmole, 10 μCi/μl) (PerkinElmer, catalog number: BLU502A )
- 3 M NaOAc (pH 4.7) (see Recipes)
Equipment
- IllustraTM MicroSpinTM G-25 column (GE Healthcare, model: 27-5325-01 )
Procedure
文章信息
版权信息
© 2012 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Putzer, H. (2012). Synthesis of 5’ end-labeled RNA. Bio-protocol 2(14): e241. DOI: 10.21769/BioProtoc.241.
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分类
分子生物学 > RNA > RNA 合成
分子生物学 > RNA > RNA 标记
分子生物学 > RNA > 转录
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