Invitro RNA synthesis from plasmid-borne sequences

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.