T7 RNA Polymerase
Temporarily discontinued!
T7 RNA polymerase is a highly specific DNA dependent RNA polymerase originated from T7 bacteriophage. One of the most potent tools used for the study of RNA dynamics.
(SKU: RN003)
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T7 RNA polymerase is a highly specific DNA dependent RNA polymerase originated from T7 bacteriophage. One of the most potent tools used for the study of RNA dynamics.
Applications
- Cell free production of proteins (in vitro transcription – translation)
- Radiolabeled probe preparation for DNA and RNA blots
- Biochemical studies of RNA molecules (structure, processing, binding)
- Knock down or Knock out of genes (siRNAs or sgRNAs production respectively)
- In vitro therapeutics (anti-cancer therapies, mRNA vaccines etc.)
Transcription Guidelines
- Standard reaction conditions:
200U T7 Polymerase | 2μl |
10x T7 Buffer | 2μl |
DNA substrate* | 1 μg** |
RNTPs mix (40 mM) | 2 μl |
RNase inhibitor | 20 u |
Sterile ultrapure water | Up to 20 μl |
Incubate for 120 min at 37oC |
*PCR products, annealed oligos or digested plasmid containing T7 promoter Sequence: TAATACGACTCACTATA, ** 0,1 to 2 μg of template DNA have been successfully tested yielding on average 10-40 times of the RNA amount eg. 15 μg of RNA were produced from 250 ng of pre annealed DNA oligos containing T7 RNA promoter.
Setup guidelines:
Make sure that buffer has been thawed completely (no white precipitant due to DTT precipitation is visible). Thaw well at room temperature. Add reactants with the following order : water, buffer (mix well) , RNAse inhibitor, template (mix well), RNTPs, T7 RNA polymerase.
- Length of transcripts. 200 units of T7 RNA polymerase have successfully produced transcripts ranging from 120 -6000 nucleotides. Expected full length transcripts were found to be above 95% pure based on an ethidium bromide-stained agarose gel
- 10-40 times the amount (mass) of input DNA template. If more quantity is needed scale up according to the standard reaction
Provided with 10 X T7 RNA pol buffer: 400 mM Tris-HCl (pH 7.9 @ 25°C) 80 mM MgCl2 10mM DTT 20mM spermidine.
Inactivation: Heat inactivation at 70oC for 20 minutes or by the addition of EDTA at a final of 5 mM (not recommended when EDTA may inhibit downstream applications)
Storage conditions:
- 100 mM NaCl
- 50 mM Tris-HCl (pH 7.9 @ 25°C)
- 20 mM β-ME
- 1 mM EDTA
- 50% Glycerol
- 0.1% w/v Triton X-100
Non-specific nuclease activity: 200 units of T7 RNA polymerase do not produce any unspecific cleavage products after 6 hrs incubation with neither 1 μg of Lambda DNA nor pBR322 at 37oC .
No RNASE activity: 200 units of T7 RNA polymerase do not produce any degradation after 6 hrs incubation with total RNA from HeLa cells.