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
----
10µl

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

Spin 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)

Locate the band by exposing the gel to film

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.

Notes:

The cap structure can be altered by replacing cap analog with e.g. GMP, GpppG, or leaving it out altogether
It’s important to set up the reaction at room temp because the spermidine in the buffer will precipitate on ice
Cold transcriptions can be set up similarly – just use 5mM UTP instead of 0.5mM
DNA template should be linearized prior to use. SP6 doesn’t like 3’-overhangs so select your restriction enzyme accordingly.
Around half of the hot UTP should get incorporated, so if your pellet doesn’t sound hot the reaction probably didn’t work.
Gel purification serves several purposes:
- Check that the RNA is intact, the correct size and not degraded
- Remove unincorporated nucleotides
- Remove template DNA
The number of moles of RNA can be calculated using the number of cpm incorporated.
Remember you’re working with RNA – make sure all solutions are RNAse free.
Remember you’re working with radioactivity – monitor for contamination after you’re done, make sure all waste is disposed of appropriately – and fill in the @%$&*! book

Troubleshooting:

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.