Knight:In vitro transcription

Protocol in progress. Use at your own risk.

Purpose

In vitro transcription using Escherichia coli RNA polymerase.

Materials

• E. coli RNA Polymerase Sigma-Saturated Holoenzyme from Epicentre - (protocol)
• Linearized template DNA
• NTPs
• DTT

Procedure

Prepare template DNA

1. 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.)

Option 1: Preincubate repressor and DNA

Preincubate repressor with template DNA (optional)

1. Mix
   • 20 μL repressor
   • 5 μL of PCR template <-perhaps cut this down? DNA is a pretty bright band?
     • Do the same for relevant controls.
2. Incubate 2 hours on benchtop.
3. Make up 50 μL reaction
   • 25 μL repressor-DNA mixture
   • 10 μL 5X E. coli RNA polymerase transcription buffer
   • 0.5 μL of 500 mM TCEP since DTT chelates zinc
   • 10 μL of 2.5 mM each NTP
   • 2 μL RNase free H₂O
   • 2.5 μL E. coli RNA polymerase holoenzyme

Incubate at 37°C for 1 hr.

Option 2: Set up transcription reaction

1. Make up 50 μL reaction
   • 22 μL RNase free H₂O
   • 10 μL 5X E. coli RNA polymerase transcription buffer
   • 5 μL of 100 mM DTT <-- replace with 0.5 μL of 500 mM TCEP since DTT chelates zinc
   • 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
2. Incubate at 37°C for 1 hr.

DNase treatment (optional)

An optional step is to treat the reaction with RNase free DNaseI to remove the template DNA.

1. Add 6 μL DNaseI buffer
2. Add 3 μL H₂O
3. Add 1μL DNaseI
4. Incubate 1 hr at 37°C
5. Heat inactivate for 10 mins at 75°C

Notes

• 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.

Notes

1. 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.
2. 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. [1]

References

1. 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)