Knight:In vitro transcription

Purpose

In vitro transcription using Escherichia coli RNA polymerase.

Materials

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

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.)

Set up reaction

From Epicentre.

Functional test

50 μL reaction

• 1X E. coli RNA polymerase transcription buffer
  • 0.04 M Tris-HCl (pH 7.5)
  • 0.15 M KCl
  • 10 mM MgCl₂
  • 0.01% Triton® X-100
• 2mM DTT
• 0.25mM NTPs
• 1μg template

Assay test

50 μL reaction

• 1X E. coli RNA polymerase transcription buffer
  • 0.04 M Tris-HCl (pH 7.5)
  • 0.15 M KCl
  • 10 mM MgCl₂
  • 0.01% Triton® X-100
• 10 mM DTT
• 0.5 mM NTPs
• 1μg template

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)