Knight:In vitro transcription: Difference between revisions
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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 18:50, 6 December 2006
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
- 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.)
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.