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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 H2O 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.
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cDNA Synthesis 