In Vitro Transcription Protocol
Hot transcription reactions
Assemble the following in order at room temperature:
1µl DNA template (~ 1µg plasmid DNA or ~ 100ng PCR product)
1µl 10x RNA pol buffer
1µl capping NTPs (5mM ATP, CTP, 0.5mM GTP, UTP)
1µl cap analog (5mM)
4.5µl a- 32P-UTP (800Ci/mmol)
0.5µl RNAse Inhibitor
1µl SP6 polymerase
Incubate 37 ˚C, 1 hour (up to 3 hours is ok).
Add 90µl dH2O
Phenol CHCl3 extract
Add 33µl 10M Ammonium Acetate
Add 250µl 100% ethanol
Precipitate at -80˚C for 10min
Wash the pellet with 70% ethanol
Dry, resuspend in 10µl RNA loading dye
Heat 30 sec
Load on 5% denaturing acrylamide gel (pre-run @600V)
Excise and elute o/n in 400µl HSCB.
Next day, phenol/CHCl3 extract, EtOH ppt (no need to add salt as there’s plenty in the HSCB), resuspend in 21µl H2O and count 1µl – a good transcription will give you >5x106cpm total.
Dilute to 100kcpm/µl for use in deadenylation assays etc.
Poor incorporation is usually due to poor quality template. Template can be treated with proteinase K and phenol extracted prior to use. DNA should be Qiagen or PEG prepped.
RNA product is too large. Usually due to incomplete digestion of the template DNA – the polymerase will just run around the plasmid making a large MW product.
Multiple RNA products. RNA is degraded – make sure solutions are RNAse free. Some templates will repeatedly give two or more products. This can be due to secondary structure – just cut out the product that’s the expected size – usually the largest one.