SP6 RNA Polymerase

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.