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.