N.B: 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: 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 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/μl prior to use. 4) The standard reaction conditions are set up containing the following quantities: 2μl (400ng) linearized plasmid DNA 20U ribonuclease inhibitor 2μl 100mM DTT 4μl 5 x T3/T7 RNA polymerase buffer 4μl 2.5mM NTPs Sterile, DEPC treated H2 O to a final volume of 19.5ul 25U (0.5ul) T3/T7 RNA polymerase The reaction mixture is incubated at 37℃ 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℃. The reaction mixture is phenol/chloroform extracted twice, ethanol precipitated, vacuum dried and resuspended in 25μl sterile, DEPC-treated TE, pH7.5. 6) RNA can be analysed be electrophoresis using denaturing conditions in an agarose/formaldehyde gel system. |
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