Riboprobe by in vitro transcription
- 최초 등록일
- 2014.09.13
- 최종 저작일
- 2014.08
- 4페이지/
한컴오피스
- 가격 1,000원
![할인쿠폰받기](/images/v4/document/ico_det_coupon.gif)
목차
1. INTRODUCTION
2. MATERIALS
3. METHOD
4. REFERENCES
본문내용
INTRODUCTION
In vitro transcription of the synthesis of RNA from a DNA template using a bacteriophage RNA polymerase. This protocol describes a procedure for synthesizing RNA probes of high specific activity (1 x 109 dpm/ug) from double-stranded linear DNAs containing promoters for bacteriophage-encoded RNA polymerases. IMPORTANT Prepare all reagents used in this protocol with DEPC-treated H2O.
In Vitro Synthesis of RNA by Bacteriophage-encoded RNA Polymerases
The DNA sequence to be transcribed is cloned into a restriction site in a polycloning region that is flanked by promoters for two bacteriophage polymerases (usually T7 and SP6). The DNA is then linearized with a restriction enzyme that cleaves downstream from the region to be transcribed. This cleavage presents synthesis of multimeric transcripts from circular template DNA. Transcription begins from the promoter proximal to the insert when the appropriate polymerase and rNTPs are added in suitable buffer. The RNA products, which are complementary to one strand of the template, are equal in length to the distance between the active prompter and the end of the linear DNA distal to the target sequences. At the end of the reaction, the template is removed by digestion with RNase-free DNase. Unincorporated nucleotides and oligodeoxynucleotides produced by digestion of the template DNA with DNase can be removed by chromatography through Sephadex G-75.
참고 자료
Krieg P.A. and Melton D.A. 1984. Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res. 12:7057-7070.
Melton D.A., Krieg P.A., Rebagliati M.R., Maniatis T., Zinn K., and Green M.R. 1984. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12:7035-7056.
Sambrook and Russel. Chapter 9: Preparation of Radiolabeled DNA and RNA probe: Molecular Cloning. 3rd Ed. New York: Cold Spring Harbor, Vol 2: 9.29-9.37