Difference between revisions of "Knight:In vitro transcription"

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#Generate linearized template via either PCR or restriction digest.   
 
#Generate linearized template via either PCR or restriction digest.   
 
#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.)
 
#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===
 
===Set up reaction===
From Epicentre
+
From Epicentre.
  
 
====Functional test====
 
====Functional test====

Revision as of 17:50, 6 December 2006

Purpose

In vitro transcription using Escherichia coli RNA polymerase.

Materials

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.