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 either PCR or restriction digest.
2. Ambion recommends a Proteinase K treatment followed by a phenol:chloroform extraction to eliminate all traces of RNase prior to subsequent reactions. (Note that if the DNA template has been linearized by restriction enzyme digestion, a similar treatment is recommended, since restriction enzymes may be contaminated with RNases.)

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.