Knight:In vitro transcription

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(Materials)
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*Linearized template DNA
*Linearized template DNA
*NTPs
*NTPs
 +
*DTT
==Procedure==
==Procedure==
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===Set up reaction===
===Set up reaction===
-
From Epicentre.
 
-
 
-
====Functional test====
 
50 μL reaction
50 μL reaction
-
*1X ''E. coli'' RNA polymerase transcription buffer
+
*10 μL 5X ''E. coli'' RNA polymerase transcription buffer
-
**0.04 M Tris-HCl (pH 7.5)
+
*0.5 μL of 1M DTT
-
**0.15 M KCl
+
*10 μL of 2.5 mM each NTP
-
**10 mM MgCl<sub>2</sub>
+
*5 &mu;L of PCR template
-
**0.01% Triton® X-100
+
-
*2mM DTT
+
-
*0.25mM NTPs
+
-
*1&mu;g template
+
-
====Assay test====
+
Incubate at 37&deg;C for 1 hr.
-
50 &mu;L reaction
+
-
*1X ''E. coli'' RNA polymerase transcription buffer
+
-
**0.04 M Tris-HCl (pH 7.5)
+
-
**0.15 M KCl
+
-
**10 mM MgCl<sub>2</sub>
+
-
**0.01% Triton® X-100
+
-
*10 mM DTT
+
-
*0.5 mM NTPs
+
-
*1&mu;g template
+
===Analyze transcription products===
===Analyze transcription products===

Revision as of 12:21, 12 December 2006

Contents

Purpose

In vitro transcription using Escherichia coli RNA polymerase.

Materials

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

50 μL reaction

  • 10 μL 5X E. coli RNA polymerase transcription buffer
  • 0.5 μL of 1M DTT
  • 10 μL of 2.5 mM each NTP
  • 5 μL of PCR template

Incubate at 37°C for 1 hr.

Analyze transcription products

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) [AmbionTemplate]
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