涂7体外转录翻译PPT.ppt

In vitro transcription and In vitro translation;In vitro transcription;In Vitro Transcription;In vitro Transcription;;Vector Maps;In vitro Transcription;Gloves should be worn at all times during preparation of in vitro RNA. All solutions should be RNase-free i.e. made with DEPC water if home-made or bought specifically to use for RNA work. ;You will need: Ribonuclease inhibitor (RNasin)T7 or T3 RNA Polymerase 2.5mM rNTPs5 x T7/T3 RNA Polymerase Buffer (200mM Tris.Cl pH 8.0, 125mM NaCl, 40mM MgCl2, 10mM spermidine) 100mM DTT RNase-free DNase I DEPC-treated, autoclaved;Linearize the plasmid at, or near, the terminus of the insert cDNA with a suitable restriction endonuclease. 2) Extract plasmid DNA with an equal volume of phenol/chloroform and an equal volume of chloroform prior to ethanol precipitation. 3) Linearized plasmid DNA is resuspended in DEPC-treated, autoclaved TE, pH7.5 at a concentration of approximately 200ng/ul prior to use. ;4) The standard reaction conditions are set up containing the following quantities: 2ul (400ng) linearized plasmid DNA20U ribonuclease inhibitor2ul 100mM DTT4ul 5 x T3/T7 RNA polymerase buffer4ul 2.5mM NTPsSterile, DEPC treated H2O to a final volume of 19.5ul25U (0.5ul) T3/T7 RNA polymerase The reaction mixture is incubated at 37°C for 60 minutes. ;5) DNA template is removed by the addition of 25U RNase-free DNase I and a further incubation for 30 minutes at 37°C. The reaction mixture is phenol/chloroform extracted twice, ethanol precipitated, vacuum dried and resuspended in 25ul sterile, DEPC-treated TE, pH7.5. 6) RNA can be analysed be electrophoresis using denaturing conditions in an agarose/formaldehyde gel system. ;In Vitro Transcription;Advantages of RNA probes: RNA-RNA hybrids are very stable Tissue can be digested with RNase (dsRNA is not digested) after the hybridization reducing the background Higher specific activity compared to oligonucleotides;In Vitro Transcribed siRNAs;;;In vitro translation;测序技术,