Tuesday, November 18, 2003
Description In vitro RNA synthesis from plasmid-borne sequences under the control of T phage promoters Procedure 1) 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 DNA 20U ribonuclease inhibitor 2ul 100mM DTT 4ul 5 x T3/T7 RNA polymerase buffer 4ul 2.5mM NTPs Sterile, DEPC treated H2O to a final volume of 19.5ul 25U (0.5ul) T3/T7 RNA polymerase The reaction mixture is incubated at 37C 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 37C. 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. Recipes 100mM DTT (Gibco-BRL) Ribonuclease inhibitor (RNasin, Pharmacia) T7 or T3 RNA Polymerase (Gibco-BRL) 2.5mM rNTPs (Promega) 5 x T7/T3 RNA Polymerase Buffer (200mM Tris.Cl pH 8.0, 125mM NaCl, 40mM MgCl2, 10mM spermidine, Gibco-BRL) RNase-free DNase I (Pharmacia) DEPC-treated, autoclaved, nano-pure water Supplies Tips (责任编辑:泉水) |