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 homemade 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.
D. In vitro transcription
1.
Prepare the following mix (from Promega's Gemini II kit)
Transcription buffer
20 ul
100 mM DTT
10 ul
rRNasin
2.5 ul
rATP/rCTP/rGTP/rUTP
5 ul each
IS PCR product
45 ul
T7 RNA polymerase
2 ul
Incubate for 1-2 hr at 37 C
2. Add 2 ul RQ1 RNase-Free Dnase. Incubate for 15-30 min at 37 C.
3. Add 100 ul TE-buffered phenol. Vortex for 1 min and centrifuge at
12,000 g for 10 min.
4. Transfer upper phase to a fresh tube. Add 100 ul chloroform/isoamyl
alcohol (24:1).
5. Transfer upper phase to a fresh tube. Add 50 ul 10 N ammonium acetate
(pH 4.0) and 500 ul ethanol. Precipitate at -20 C for 30 min.
6. Spin at 12,000 g for 10 min. Wash with 70% ethanol.
7. Quantitate RNA using absorbance at 260 nm. [Note: to quantitate RNA
use the following formula: ABS260 x 0.04 x dilution factor = ug/ul].
8. How to calculate molecules/ul of IS:
ug/ul
____________________________________
(330 ug/umol/bp x bp IS)
x 6.02 E 17 mlcls/umole
The 330 x bp is an approximation for the molecular weight of the internal
standard. For example, a 0.1 mg/ml solution of a 400 bp IS would be 4.56
x 1011 mlcls/ul.