Description
T7 RNA Polymerase catalyses the synthesis of RNA in the presence of double-stranded DNA containing a T7 promoter sequence. It is isolated from an E. coli transformed by a plasmid containing the T7 RNA Polymerase gene. This enzyme is provided in trehalose-based storage buffer containing: 20 mM potassium phosphate pH 7.5, 100 mM NaCl, 1.0 mM EDTA, 10 mM DTT, 0.2% Triton X-100 and 50% glycerol. Alternatively, it is also provided in the same buffer containing 1.0 M trehalose instead of glycerol (suitable for lyophilization). Concentration 70,000 units/mL.
Please inquire for custom concentrations and bulk quantities.
Applications:
Preparation of fluorescent or radioactively labeled RNA probes
Preparation of large RNA transcripts for analysis or in vitro translation
Amplification of RNA (in conjunction with AMV RT and Ribonuclease H.)
Preparation of anti-sense RNA
Preparation of ribozymes
Preparation of mRNA precursor for splicing
Preparation of ds RNA for RNA interference or silencing
Preparation of substrate in RNase protection assays
Preparation of template for genomic DNA sequencing
Preparation of RNA for studies of RNA secondary structure and RNA-protein interactions
Reagents supplied:
5X Reaction Buffer for T7 RNA Polymerase
1X Reaction Buffer Conditions:
40 mM TrisHCl, pH 7.7
6 mM MgCl2
10 mM DTT
2 mM Spermidine
To be supplemented with:
0.5 mM ATP, CTP, GTP and UTP
DNA template with T7 promoter
(20 nM of a linearized plasmid DNA or 40 ug/mL of a 3 Kb plasmid)
Incubate at 37ºC
Unit Definition:
One unit is defined as the amount of enzyme that will incorporate 3 nmol of Uridine triphosphate into acid-insoluble form in one hour at 37ºC under standard assay conditions: 40 mM TrisHCl, pH 7.5, 30 mM MgCl2, 20 mM DTT, 0.4 mM GTP, CTP,ATP, UTP and
[3H]-UTP, 20 µg/mL non-linearized plasmid DNA, 50 µg/mL BSA.
General Notes:
Dithiothreitol is required for T7 RNA Polymerase activity. This labile component of the reaction buffer may be restored by supplementing reactions with a final concentration of 10 mM DTT.
Higher yields of RNA may be obtained by increasing the concentration of NTP’s to as much as 4 mM.
Care must be taken that the total salt concentration in the reaction does not exceed 50mM, since this enzyme is sensitive to salt concentrations exceeding this amount.
Quality Control of T7 RNA Polymerase
1.) DNase Activity: One-half µg of Hae fragments of Phi X-174 DNA is incubated at 37ºC with 175 units of T7 RNA Polymerase for 3 hours, and then electrophoresed in a native agarose gel simultaneously with control positive DNase 1 reactions. No more than the equivalent of 1.25 X 10E-4 unit of DNase 1 is detected.
2.) Ribonuclease Activity: One microgram of an RNA Ladder is incubated for 2 hours with 280 units of T7 RNA Polymerase, and then electrophoresed in a native agarose gel simultaneously with control positive RNase 1A reactions. No more than the equivalent of 8.0 X 10E-8 unit of RNase 1A is detected.
3.) Specific Activity: The specific activity of the T7 RNA Polymerase is no less than 400,000 units per mg.
References:
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, (2nd Ed.), 10.27-10.37
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, (2nd Ed.), 18.82-18.84
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