T7 transcription kit with modified T7 RNA Polymerase for higher tolerance towards modified nucleotides. Extremely useful for radioactive and nonradioactive labeling as well as for RNA synthesis in preparative scale.
Description:
- DNA-dependent RNA polymerase with stringent specificity for T7 phage promoters sequence (1).
- Ultrapure recombinant enzyme.
- Efficiently synthesizes in vitro transcripts from almost any DNA target located downstream from a T7 promoter (2).
- Suitable for preparing labeled single-stranded RNA probes of high specific activity (3).
- Transcripts can be used as hybridization probes, templates for in vitro translation, substrates in RNA processing systems, as well as for exon and intron mapping of genomic DNA.
Unit Definition: One unit is the amount of enzyme required to incorporate 1 nmol of labeled UTP into acid-soluble material in 1 hr at 37°C.
Storage Conditions: Store at -20°C
Storage Buffer: 20 mM potassium phosphate (pH 7.7), 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol and 50 % (v/v) glycerol.
Quality Control: All preparations are assayed for contaminating exonuclease, endonuclease , for nonspecific RNase and single- and double-stranded DNase activities. Typical preparations are greater than 90 % pure, as judged by SDS polyacrylamide gel electrophoresis.
References:
- Chamberlin, M. and Ring, J. (1973) J. Biol. Chem. 248, 2235-2244.
- Tabor, S and Richardson, C.C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1074-1078.
- Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, second edition, pp. 10.27-10.37, Cold Spring Harbour Laboratory, Cold Spring Harbour.
Kit component: 25 rxn: 50 rxn:
5 x T7 Reaction Buffer 150 µl 300 µl
NTPs mix 25 mM each 50 µl 100 µl
DTT 100 mM 50 µl 100 µl
Thermophilic Pyrophosphatase 2.5 U/µl 12.5 µl 25 µl
T7 RNA Polymerase 12.5 µl 25 µl
RNase-free water 1 ml 1 ml
RNA loading buffer 50 µl 100 µl
In vitro T7 transcription 25 µl:
Reaction assembly should be performed at room temperature (not on ice). This prevents any precipitation of template DNA due to spermidine contained in the 5 x T7 Reaction Buffer.
5 µl 5 x T7 Reaction Buffer
1.8 µl NTPs mix 25 mM each
1.25 µl DTT 100 mM
0.5 µl Thermophilic Pyrophosphatase 2.5 U/µl (EURx Cat. No. E1267)
1-2 µg DNA template*
0.5 µl T7 RNA Polymerase**
to 25 µl RNase-free water
Incubate up to 2 hours at 37°C, then check transcription on appropriate denaturing polyacrylamide gel. Load 5 µl of reaction mixed with 3 µl of RNA loading buffer.
*High purity of the template is very important for the yield of reaction. If run off transcription is applied be sure there is no RNase A contamination that could be due to plasmid preparation. We recommend using our RNase free Plasmid DNA Purification kit (Cat. No. E3500), which works excellent for preparing RNase free plasmid DNA. In case, T7 template DNA is a PCR fragment, remove primers (recommended: Purification from Agarose Gels using e.g. our Agarose-out DNA Purification Kit, Cat. No. E3540) and confirm DNA homogeneity on an agarose gel.
**0.2 µl of T7 RNA polymerase is most efficient for labeling, more enzyme is recommended for preparative
lamide gel. Load 5 µl of reaction mixed with 3 µl of RNA loading buffer.
*High purity of the template is very important for the yield of reaction. If run off transcription is applied be sure there is no RNase A contamination that could be due to plasmid preparation. We recommend using our RNase free Plasmid DNA Purification kit (Cat. No. E3500), which works excellent for preparing RNase free plasmid DNA. In case, T7 template DNA is a PCR fragment, remove primers (recommended: Purification from Agarose Gels using e.g. our Agarose-out DNA Purification Kit, Cat. No. E3540) and confirm DNA homogeneity on an agarose gel.
**0.2 µl of T7 RNA polymerase is most efficient for labeling, more enzyme is recommended for preparative scale.
Description:
- DNA-dependent RNA polymerase with stringent specificity for T7 phage promoters sequence (1).
- Ultrapure recombinant enzyme.
- Efficiently synthesizes in vitro transcripts from almost any DNA target located downstream from a T7 promoter (2).
- Suitable for preparing labeled single-stranded RNA probes of high specific activity (3).
- Transcripts can be used as hybridization probes, templates for in vitro translation, substrates in RNA processing systems, as well as for exon and intron mapping of genomic DNA.
Unit Definition: One unit is the amount of enzyme required to incorporate 1 nmol of labeled UTP into acid-soluble material in 1 hr at 37°C.
Storage Conditions: Store at -20°C
Storage Buffer: 20 mM potassium phosphate (pH 7.7), 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol and 50 % (v/v) glycerol.
Quality Control: All preparations are assayed for contaminating exonuclease, endonuclease , for nonspecific RNase and single- and double-stranded DNase activities. Typical preparations are greater than 90 % pure, as judged by SDS polyacrylamide gel electrophoresis.
References:
- Chamberlin, M. and Ring, J. (1973) J. Biol. Chem. 248, 2235-2244.
- Tabor, S and Richardson, C.C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1074-1078.
- Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, second edition, pp. 10.27-10.37, Cold Spring Harbour Laboratory, Cold Spring Harbour.
Kit component: 25 rxn: 50 rxn:
5 x T7 Reaction Buffer 150 µl 300 µl
NTPs mix 25 mM each 50 µl 100 µl
DTT 100 mM 50 µl 100 µl
Thermophilic Pyrophosphatase 2.5 U/µl 12.5 µl 25 µl
T7 RNA Polymerase 12.5 µl 25 µl
RNase-free water 1 ml 1 ml
RNA loading buffer 50 µl 100 µl
In vitro T7 transcription 25 µl:
Reaction assembly should be performed at room temperature (not on ice). This prevents any precipitation of template DNA due to spermidine contained in the 5 x T7 Reaction Buffer.
5 µl 5 x T7 Reaction Buffer
1.8 µl NTPs mix 25 mM each
1.25 µl DTT 100 mM
0.5 µl Thermophilic Pyrophosphatase 2.5 U/µl (EURx Cat. No. E1267)
1-2 µg DNA template*
0.5 µl T7 RNA Polymerase**
to 25 µl RNase-free water
Incubate up to 2 hours at 37°C, then check transcription on appropriate denaturing polyacrylamide gel. Load 5 µl of reaction mixed with 3 µl of RNA loading buffer.
*High purity of the template is very important for the yield of reaction. If run off transcription is applied be sure there is no RNase A contamination that could be due to plasmid preparation. We recommend using our RNase free Plasmid DNA Purification kit (Cat. No. E3500), which works excellent for preparing RNase free plasmid DNA. In case, T7 template DNA is a PCR fragment, remove primers (recommended: Purification from Agarose Gels using e.g. our Agarose-out DNA Purification Kit, Cat. No. E3540) and confirm DNA homogeneity on an agarose gel.
**0.2 µl of T7 RNA polymerase is most efficient for labeling, more enzyme is recommended for preparative scale.