5'末端标记RNA的合成

Harald Putzer

doi:10.21769/BioProtoc.241

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Peer-reviewed

Synthesis of 5’ end-labeled RNA

5'末端标记RNA的合成

Harald Putzer email

发布: 2012年07月20日第2卷第14期 DOI: 10.21769/BioProtoc.241 浏览次数: 14215

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参见作者原研究论文

The authors used this protocol in:

Cover of Molecular Microbiology, featuring study using the protocol.

Dec 2011

Special Issue on Protocols for Organoid Research

实验方案合集

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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.

Keywords: RNA Labeling (RNA标记)

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)