MicroRNA和siRNA克隆规程（MicroRNA and siRNA Cloning Protocol）

RNA Cloning Method Flowchart

 

Extract total RNA containing small RNAs. Check quality on denaturing gel with EtBr staining

5" End label RNA markers 18.113 (18mer) and 44.12 (24mer) with Kinase and 32 P-gamma-ATP. Gel purify labeled markers.

Electrophorese total RNA with radiolabeled RNA markers. Visualize by phosphorimaging. Cut out gel slice containing both RNA markers. Elute RNA, precipitate, and resuspend.

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Ligate App17.91x to the 3" ends of gel-purified small RNA pool. Electrophorese reaction on denaturing gel. Gel purify ligated products by following shifted mobility of RNA markers.

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Ligate 17.93R to the 5" ends of gel-purified RNAs. Electrophorese reaction on denaturing gel. Gel purify ligated products by following shifted mobility of RNA markers.

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Reverse transcribe small RNA pool with 15.22.PCR amplify with 17.92 and 17.93D for 15-25 cycles. Purify PCR-amplified pool by phenol extraction/EtOH precipitation. Digest pool with Ban-I.

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Phenol extract/EtOH precipitate restriction digest reaction. Concatamerize DNA fragments with T4 DNA ligase. Gel purify concatamers from agarose gel with classical phenol extraction method.

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Screen colonies for vectors containing cloned concatamers by PCR with M13F and M13R primers. Purify positive PCR products or plasmids for sequencing. Submit to sequencing service.

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Manually inspect chromatograms.Analyze sequences manually or with automated algorithms

OLIGOS FOR SMALL RNA CLONING

Underline nucleotides mark a Ban I restriction digest site (G↓GYRCC)
3" End Donor Oligo (available from IDT Inc. as the miRNA cloning linker)
App17.91x: AppCTGTA GGCACC ATCAddA
Note: To synthesize this oligo in your lab, see the original 2001 version of the cloning prot℃ol on the Bartel Lab website.
5" End Acceptor Oligo (RNA/DNA version, lowercase RNA)
17.93R: ATCGTa ggcacc ugaaa
3" RT Primer Oligo (Shorter 5" end to minimize mispriming)