In vitro RNA synthesis from plasmid-borne sequences under the control of T phage prom

 

In vitro RNA synthesis from plasmid-borne sequences under the control of T phage promoters

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/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 H₂ O to a final volume of 19.5ul
25U (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.