RNA标记
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1904
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RNA labeling (Amersham Pharmacia)
For the generation of radiolabelled, single stranded RNA for use as hybridization probes
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32 P-pCp 3' End-labeling RNA (Jim Brown Lab)
This procedure includes two methods for the synthesis of 32P-pCp, because of the enormous cost of the commercial product (about $1 per uCi). The compound is easy to synthesis, & is fresher than the commercial product.
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RNA end-labeling (gopher://iubio.bio.indiana.edu )
RNA can be 5' or 3' end-labeled. 3' end-labeling is carried out by RNA ligase, which 'ligates' a single labeled nucleotide to the 3'-OH end of the RNA. 5' end-labeling is carried out exactly as for DNA (see that procedure in M&M as well), unless the 5' ends are capped; in that case, the RNA must first be de-capped with tobacco acid pyrophosphatase (TAP).and 5' labeling
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Digoxigenin-UTP RNA Labeling (Frank Bottone Jr.)
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Preparing 5' end-labeled alkaline hydrolyzed RNA (Jim Brown Lab)
The best way to obtain high specific activity labeled rRNAs is to hydrolyze the RNA by alkaline treatment at high temperature, followed by 5' end-labeling with PNK & gamma-32P-ATP. This way, you get many 32P's incorporated into each rRNA molecule. This is also useful in heterologous probings, because the similarities of some of the RNA fragments will be much higher than the overall similarity for the heterologous rRNAs.
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Sephadex G-50 Spin Column (gopher://iubio.bio.indiana.edu )
For purifying labeled probe
