In vitro transcription

IN VITRO TRANSCRIPTION

Last Update: December 2006

PROCEDURE - IN VITRO TRANSCRIPTION

1. Set up reaction: Mix 1 mL of linearized DNA template pBS-SK- ERD (X-E) (0.1-0.5 mg), 7.8 mL of dH₂O, 5 mL of Stratagene 5X transcription buffer, 1 mL of 750mM DTT, 1 mL of Rnasin (RNase inhibitor; 15U/ml), 1 mL of 10mM ATP (pH 7), 1 mL of 10mM CTP (pH 7), 1 mL of 10mM GTP (pH 7), 1 mL of 1 mM UTP (pH 7), 5 mL of (a-³²P )-UTP (400-800 Ci/mmol; 10 mCi/mL) (final conc. 4-8mM), 0.2 mL T7 or T3 or SP6 RNA polymerase (50 U/mL, Stratagene). Total volume: 25 mL

2. Mix, spin, and incubate at 37^oC for 30 minutes

3. Add 1 mL of DNase 1 (free from Rnase -10 U/mL)

4. Mix, spin, and incubate at 37^oC for 15 minutes

5. End the reaction by adding: 7 mL of 10X STE (0.2 M Tris-HCl, pH 7.5; 1 M NaCl; 0.1 M EDTA) and 38 mL of dH₂O

6. Mix by pipeting and spot 1 mL on paper disk inside a scintillation bottle

7. Purify the rest of the probe through a gel filtration PUSH COLUMN (Stratagene)

PROCEDURE - Purification of RNA probe away from unincorporated radiolabelled nucleotides using push gel filtration column

1. Wet Stratagene push column (#400701, 400700) with 80 mL of STE buffer (20 mM Tris-HCl, pH 7.5; 0.1 M NaCl; 10 mM EDTA)

2. Load thetranscription reaction on the column. Place a screw cap tube at the end to collect the radioactive probe. Pipette on top of the column 70 mL more STE and collect into the same tube. Mix and measure volume (~110-130 mL)

3. Calculate radioactive incorporation

3.1 Spot 1 mL of the eluted probe on paper

3.2 Add to paper disk 4 mL of scintillation cocktail and read in scintillation counter

3.3 (B) = CPM/1 x 70 mL = B - CPM

3.4 (A) = CPM /1 x Elution Volume (mL) = A - CPM

3.5 A-CPM/B-CPM x 100 = % incorporation (Usually 40-50% incorporation)

3.6 specific activity - usually 1-5 X10⁹ cpm/mg RNA

4. The probe is ready for hybridization