Knight:In vitro transcription
Protocol in progress. Use at your own risk.
In vitro transcription using Escherichia coli RNA polymerase.
- E. coli RNA Polymerase Sigma-Saturated Holoenzyme from Epicentre - (protocol)
- Linearized template DNA
Prepare template DNA
- Generate linearized template via PCR. Do a 100 μL reaction using VF2 and VR. Use 5 μL directly in the trancription reaction. (Is this enough DNA? Epicentre recommends 1 μg.)
Set up transcription reaction
50 μL reaction
- 10 μL 5X E. coli RNA polymerase transcription buffer
- 5 μL of 100 mM DTT
- 10 μL of 2.5 mM each NTP
- 5 μL of PCR template <-perhaps cut this down? DNA is a pretty bright band?
- 2.5 μL E. coli RNA polymerase holoenzyme
- 22 μL RNase free H2O
Incubate at 37°C for 1 hr.
An optional step is to treat the reaction with RNase free DNaseI to remove the template DNA.
- Add 6 μL DNaseI buffer
- Add 3 μL H2O
- Add 1μL DNaseI
- Incubate 1 hr at 37°C
- Heat inactivate for 10 mins at 75°C
- EDTA should be added to a final concentration of 5 mM to protect RNA from being degraded during enzyme inactivation. But EDTA can chelate magnesium which is needed for DNaseI activity. So the EDTA may need to be added just prior to inactivation.
- DNase I is not active on DNA bound to proteins.
Analyze transcription products
Then analyze via native agarose gel electrophoresis.
- If using either plasmid DNA or DNA template has been linearized by restriction enzyme digestion, Ambion recommends a Proteinase K treatment followed by a phenol:chloroform extraction to eliminate all traces of RNase prior to subsequent reactions. This treatment is necessary because most plasmid DNA has been subjected to RNaseA during purification and restriction enzymes may be contaminated with RNases.
- However, in the case of PCR generated templates, Ambion adds amplified DNA directly to the transcription reaction with no purification. 5 μl of a 100 μl PCR reaction (or about 0.05 - 0.2 μg of double-stranded DNA) is used as template. However, with shorter templates or low yields, the concentration of template in a 5 μl aliquot of the crude PCR reaction may be suboptimal. In that case, it may be desirable to concentrate the PCR product by alcohol precipitation. We do not generally find it necessary to phenol/chloroform extract the PCR reaction before precipitation, although in some cases it may be advisable to do so. 
- A PCR Strategy for Rapid Generation of Template DNA for Synthesis of Labeled RNA Probes (contains lots of useful info about generating transcription templates via PCR)