What type of polymerase is T7?
T7 RNA Polymerase
Thermo Scientific Bacteriophage T7 RNA polymerase is a DNA-dependent RNA polymerase with strict specificity for its respective double-stranded promoters. It catalyzes the 5’→3′ synthesis of RNA on either single-stranded DNA or double-stranded DNA downstream from it promoter.
Where does T7 RNA polymerase come from?
T7 RNAP is a small (99 kDa) DNA-dependent RNAP derived from bacteriophage T7 (Chamberlin and Ring, 1973; Golomb and Chamberlin, 1974; Steitz, 2004). The polymerase efficiently and specifically transcribes genes bearing a T7 promoter (PT7).
How does T7 RNA polymerase work?
T7 RNA polymerase is a very active enzyme: it synthesizes RNA at a rate several times that of E. coli RNA polymerase and it terminates transcription less frequently; in fact, its transcription can circumnavigate a plasmid, resulting in RNA several times the plasmid length in size.
Does RNA polymerase have helicase activity?
In contrast to DNA polymerase, RNAP includes helicase activity, therefore no separate enzyme is needed to unwind DNA.
Why we use T7 RNA polymerase?
T7 RNA Polymerase is used for in vitro mRNA synthesis and is highly specific for the T7 phage promoter. Applications include: Radiolabeled RNA probe preparation. RNA generation for studies of RNA structure, processing and catalysis.
Why is a T7 promoter used?
The T7 promoter is commonly used to regulate gene expression of recombinant proteins, which can be subsequently used for a variety of downstream research applications2.
How long is T7 promoter?
The T7 promoter is a sequence of DNA 18 base pairs long up to transcription start site at +1 (5′ – TAATACGACTCACTATAG – 3′) that is recognized by T7 RNA polymerase1 .
How does the T7 expression system work?
T7 expression hosts such as DE3 prophage strains or T7 Express strains carry a chromosomal copy of the phage T7 RNA polymerase gene. When inducer is added, T7 RNA polymerase is expressed and becomes dedicated to target gene transcription.
Does RNA polymerase require ATP?
Transcription initiation by RNA polymerase II does not require hydrolysis of the beta-gamma phosphoanhydride bond of ATP. Laboratory for Physiological Chemistry, Utrecht University, The Netherlands.
Why RNA polymerase has no proofreading?
It is generally assumed that RNA pol. does not need to proofread, because RNA molecules are working copies that can tolerate a few errors (and can be replaced by new copies transcribed from the DNA).
What does T7 do to E. coli?
Once the T7 phage has inserted the viral genome, the process of DNA replication of the host genome is halted and replication of viral genome begins. Under optimal conditions, the T7 phage can complete the lytic process within 25 minutes, leading to the death of the E. coli host cell.
What induces the T7 promoter?
When IPTG is present in the medium, it will enter the cells and remove LacI from the LacO site. As a result, T7 RNA Polymerase binds to the T7 promoter and initiates gene transcription.
What is T7 RNA polymerase used for?
Thermo Scientific Bacteriophage T7 RNA polymerase is a DNA-dependent RNA polymerase with strict specificity for its respective double-stranded promoters. It catalyzes the 5’→3′ synthesis of RNA on either single-stranded DNA or double-stranded DNA downstream from it promoter. Highlights.
What is the best way to store T7 RNA polymerase?
T7 RNA Polymerase is supplied with a vial of 5X reaction buffer [200 mM Tris-HCl (pH 8.0), 40 mM MgCl 2 , 10 mM spermidine- (HCl) 3 , 125 mM NaCl], vial of 100 mM DTT. Store at -20°C. Convenient, on-site access to the products you need.
What is the T7 enzyme mix and the reaction buffer?
The T7 Enzyme Mix and the 10X Reaction Buffer are specifically calibrated for each lot and RNA polymerase. The MEGAscript T7 Kit contains in vitro transcription reaction components and a control template. The kit will yield a total of 3 to 5mg of RNA (approximately 100μg of RNA or more per reaction) from the control template supplied with the kit.
How many moles of RNA does the megascript T7 kit produce?
The MEGAscript T7 Kit contains in vitro transcription reaction components and a control template. The kit will yield a total of 3 to 5mg of RNA (approximately 100µg of RNA or more per reaction) from the control template supplied with the kit. This corresponds to 400 to 650 moles of RNA for each mole of template.