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  RNA analysis tools

T7 RNA Polymerase

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)

50.00
  • 100u/μl
  • 5000 U

Product Details

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  Polymerase2μl
10x T7 Buffer2μl
DNA substrate*1 μg**
RNTPs mix (40 mM)2 μl
RNase inhibitor20 u
Sterile ultrapure waterUp 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.

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