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SP6 RNA Polymerase |
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Prices are in US dollars and valid only for US orders.
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- Isolated from a recombinant source
- RNA probe preparation for hybridization
- mRNA generation for in vitro translation systems
- Supplied with 10X Reaction Buffer
Description:
SP6 RNA Polymerase catalyzes the synthesis of RNA in the 5´→ 3´ direction in the presence of a DNA template containing an SP6 phage promoter. SP6 RNA Polymerase can be used to generate high specific activity labeled RNA probes, RNA for in vitro translation, biologically active mRNA and/or preparative quantities of defined length RNA by run off transcription (1).
Source:
An E. coli strain that carries the cloned gene for SP6 RNA Polymerase from Salmonella typhimurium LT2Z.
Applications:- Radiolabeled RNA probe preparation (2)
- RNA generation for in vitro translation (3)
- RNA generation for studies of RNA structure, processing and catalysis (3)
- Expression control via anti-sense RNA
Reagents Supplied:
RNAPol Reaction Buffer (10X)
Reaction & Storage Conditions
Reaction Conditions:
1X RNAPol Reaction Buffer
Supplemented with 0.5 mM ATP, 0.5 mM Guanosine triphosphate, 0.5 mM UTP, and 0.5 mM CTP
Incubate at
40°C.
1X RNAPol Reaction Buffer:
40 mM Tris-HCl
6 mM MgCl2
10 mM Dithiothreitol
2 mM spermidine
pH 7.9 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme that will incorporate 1 nmol ATP into acid-insoluble material in a total reaction volume of 50 μl in 1 hour at 40°C in 1X RNA Polymerase Reaction Buffer.
Concentration:
20,000 units/ml
Storage Conditions:
50 mM Tris-HCl
100 mM NaCl
20 mM 2-Mercaptoethanol
1 mM EDTA
50% Glycerol
0.1% Triton X-100
pH 7.9 @ 25°C
Storage Temperature:
-20°C
Notes
Usage notes:- Dithiothreitol is required for activity.
- SP6 RNA Polymerase is extremely sensitive to salt inhibition. For best results, overall salt concentration should not exceed 50 mM.
- SP6 RNA Polymerase is 30% more active at 40°C than at 37°C
- Higher yields of RNA may be obtained by raising NTP concentrations (up to 4 mM each). Mg2+ concentration should be raised to 4 mM above the total NTP concentration. Additionally, inorganic pyrophosphatase should be added to a final concentration of 4 units/ml.
- An apparent decrease in enzyme activity over time may be due to the breakdown of dithiothreitol in the reaction buffer; even when stored at -20°C. If you observe a decrease in yield, try supplementing your reactions with a final concentration of 10 mM fresh dithiothreitol.
FAQs
- What is the molecular weight of SP6 RNA Polymerase?
- What are the main causes of reaction failure using SP6 RNA Polymerase?
- Is SP6 RNA Polymerase an enzyme of choice for making high specific activity labeled probes?
- Does the reaction with SP6 RNA Polymerase require a primer?
- What is the first base that SP6 RNA Polymerase transcribes?
- Does SP6 RNA Polymerase leave an extra base at the end of a transcript?
- Does SP6 RNA Polymerase require single stranded substrate?
- Will SP6 RNA Polymerase work on single stranded substrate?
- Will SP6 RNA Polymerase work on uncut plasmid DNA?
- Can aberrant RNA be produced when using SP6 RNA Polymerase?
- How can the yield of RNA be maximized when using SP6 RNA Polymerase?
- Why is the specific activity of the probe low?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
Purified free of other RNA polymerases, DNases, and RNases.
Terminal Integrity:
After incubation of 1 μg of lambda DNA (Hind III digest) with 50 units of SP6 RNA Polymerase
at 40ºC for 1 hour(s), > 95% of the DNA fragments can be ligated
with T4 DNA ligase. Of these ligated fragments, >95% can be recut with Hind III.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of λDNA
and 200 units of SP6 RNA Polymerase incubated for 16 hours at 40ºC
resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 200 units of SP6 RNA Polymerase with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 40ºC
released < 0.1% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 200 units of SP6 RNA Polymerase with 1 μg of
ΦX174 RF I DNA for 4 hours at 40ºC resulted
in < 5% conversion to RFII as determined by agarose gel electrophoresis.
RNase Assay:
Incubation of a 50 μl reaction containing 200 units of SP6 RNA Polymerase with 1
μg of RNA Ladder for 1 hour at 40ºC
resulted in no detectable degradation of the RNA as determined by agarose gel electrophoresis.
DNA Polymerase Activity:
Incubation of 200 units of SP6 RNA Polymerase in a 50 μl reaction with λ DNA as a
template resulted in < 0.1% of the amount of product incorporated when SP6 DNA was used as a template.
1 µg of control DNA template incubated with 3 units of SP6 RNA Polymerase under standard assay conditions in the absence of RNase inhibitors for 1 hour at 40°C. The reaction mixture was then treated with DNase I (to remove DNA templates), glyoxal (6) and electrophoresed on 1.8% agarose gel.
References
- Schenborn, E.T. and Meirendorf, R.C. (1985) Nucl. Acids Res., 13, 6223-6236.
- Butler, E.T. and Chamberlin, J. (1982) J. Biol. Chem., 257, 5772-5778.
- 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.
- Melton, D.A., Krieg, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K. and Green, M.R. (1984) Nucl. Acids Res., 12, 7035-7056.
- Kreig, P.A. and Melton, D.A. (1984) Nucl. Acids Res., 12, 7057-7070.
- Green, M.R., Maniatis, R. and Melton, D.A. (1983) Cell, 32, 681-694.
- Melton, D.A. (1985) Proc. Natl. Acad. Sci. USA, 82, 144-128.
- Milligan, J.F., Groebe, D.R., Witherell, G.W. and Uhlenbeck, O.C. (1987) Nucl. Acids Res., 15, 8783.
- Zinn, K. et al. (1983) Cell, 34, 865-879.
Reagents Sold Separately
RNAPol Reaction Buffer
Companion Products
dsRNA Ladder
Ribonucleotide Solution Mix
ssRNA Ladder
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