RNA analysis tools

Description

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  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 70^oC 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 37^oC .

No RNASE activity: 200 units of T7 RNA polymerase do not produce any degradation after 6 hrs incubation with total RNA from HeLa cells.