Introduction

SP6 RNA Polymerase is a DNA-dependent RNA polymerase isolated from phage-infected Salmonella typhimurium. The enzyme has an extremely high specificity for SP6 promoter sequences (1, 2) and will synthesize large quantities of RNA from a DNA fragment inserted downstream from a promoter. Strong promoter sequences have been used to construct various cloning vectors, and inserts into the multiple cloning site of these vectors can be transcribed to generate discrete RNA's.

Components:

18018-010 SP6 RNA Polymerase
Y90109 5X SP6 Buffer
Y01500 10 mM DTT

Unit Definition:

One unit incorporates 1 nmol of labeled nucleotide into acid-precipitable material in 1 hour at 37ºC.

Storage Buffer:                                      5X SP6 Buffer:
50 mM Tris-HCl (pH 7.9)                           0.2 M Tris-HCl (pH 7.9)
0.1 M NaCl                                                30 mM MgCl2
0.1 mM EDTA                                           10 mM spermidine-(HCl)3
14 mM 2-mercaptoethanol                       Refer to Functional Assay
50% (v/v) glycerol                                   Conditions on reverse side for
0.1% (w/v) Triton® X-100                       further details.
                                                                 Store buffer at -20ºC.
Store 10 mM DTT at -20ºC.

Quality Control:

This product has passed the following quality control assays: functional absence of exonuclease, endo-ribonuclease and DNA nicking activities; performance in a transcription reaction. The enclosed buffers were assayed with the enzyme and met quality control specifications.

Functional Assay Conditions:

2 μl 5X SP6 Buffer
2.5 μM [α-32P]UTP (10 μCi at 400 Ci/mmol)
10 μM UTP
0.4 mM each ATP, CTP, GTP
1 mM DTT
0.2 μg linearized template DNA
15 units SP6 RNA Polymerase
Reaction Volume:   10 μl
Incubation:   60 minutes at 37°C

NOTE: The reaction is not set up on ice due to potential precipitation of DNA in the presence of spermidine.
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References

  1. Butler, E. T., and Chamberline, M. J. (1982) J. Biol. Chem. 257, 5772.

  2. Kassavetis, G. A., Butler, E. T., Roulland, D. and Chamberlin, M. J. (1982) J. Biol. Chem. 257, 5779.


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18018-010            17-Aug-2001