MicroRNA和siRNA克隆规程（MicroRNA and siRNA Cloning Protocol）
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RNA Cloning Method Flowchart
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| 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. |
| 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. |
| 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. |
| 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. |
| 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)
