Knight:In vitro transcription

Purpose

In vitro transcription using Escherichia coli RNA polymerase.

Materials

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

Reaction conditions

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 T7 D111 DNA as 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
• 10mM DTT
• 0.5mM NTPs
• 1μg T7 D111 DNA as template