T7 RNA Polymerase
T7 RNA Polymerase is a DNA-dependent RNA polymerase from T7 phage, that possesses a strong and specific 5´→ 3´ RNA polymerase activity.T7 RNA Polymerase has high specificity for T7 promoter sequences and will synthesize large quantities of RNA from a DNA fragment inserted downstream from a promoter.
Components
T7 RNA polymerase (50 U/μL)
10×HH T7 Buffer
Storage conditions
Transportation under 0°C and storage at -25 ~ - 15 °C.
Specification
|
Product Name |
T7 RNA Polymerase |
|
Product characterization |
Clear liquid |
|
Activity unitdefinition |
One unit is defined as the amount of enzyme that will catalyze NTP to produce 1 nmol PPi in 1 hour at 37°C under the standard reaction system. |
|
Storage buffer |
50 mM Tris-HCl, 100 mM NaCl, 20 mM β-M, 1 mM EDTA, 50% Glycerol ,0. 1% (w/v) Triton® X- 100, pH 7.9 @ 25°C. |
|
10×HH T7 buffer |
400 mM Tris-HCl (25℃ , pH 8.20) ,60 mM MgCl2 ,100 mM DTT ,20 mM spermidine. |
|
Storage condition |
-25 ~ – 15℃ , avoid repeated freezing-thawing |
Reaction and Condition
Conventional reaction
|
Reagent |
Amount |
|
Nuclease-free H2O or DEPC-Treated water |
Up to 20 μL |
|
10×HH T7 Buffer |
2 μL |
|
ATP/GTP/CTP/UTP (100 mM each) |
0.4 μL each (2 mMeach Final) |
|
RNase Inhibitor (40 U/μL)（optional） |
1 μL (40 U) |
|
Pyrophosphatase Inorganic（optional） |
0.5 μL (0.05U) |
|
T7 RNA polymerase（50 U/μL） |
1 μL |
|
Linearized DNA Template |
1 μg |
Add the reaction components in the above order * 10 × HH T7 Buffer is only suitable for 2mM NTPs, other High Yield T7 transcription kits arerecommended for 7.5mM- 10mM NTPs.
Incubation Time: 37 °C for 2 hours.
Stop of Reaction: Add 2μL 0.2 M EDTA (pH8.0@25℃) or heat to 75 °C for 10min.
DNA Removal: DNA template can be removed with 2U DNase I (RNase-free) and incubation for 15 min at 37 °.
Quality control
• Endonuclease Activity: Incubation of a 50μL reaction containing a minimum of 50U of T7 RNAPolymerase with 1 μg λDNA for 16 hours at 37 ℃ results in no detectable degradation as determined.
• Exonuclease Activity: Incubation of a 50μL reaction containing a minimum of 50U of T7 RNA Polymerase with 1 μg λ -Hind Ⅲ digest DNA for 16 hours at 37℃ results in no detectable degradation as determined.
• Nickase Activity: Incubation of a 50μL reaction containing a minimum of 50U of T7 RNA Polymerase with 1μg pBR322 DNA for 16 hours at 37°C results in no detectable degradation as determined.
• RNase Activity: Incubation of a 50μL reaction containing a minimum of 50U of T7 RNA Polymerase with 1.6μg MS2 RNA for 16 hours at 37°C results in no detectable degradation as determined.
•Heat Inactivation: 75 °C for 10 min.
Precaution
• The transcription reaction should be performed in an environment free of RNase contamination. Wearing gloves is advisable. The tips, tubes and water should be nuclease free.
• The production yield of RNA can be improved by increasing the concentration of NTP (each concentration can reach 10mM), while Mg2+ concentration needs to be appropriately increased.
• The RNA synthesis reaction mixture should be prepared at room temperature, since DNA may precipitate in the presence of 10×HH T7 Buffer at 4°C.
•The yield of proper length transcripts decreases if the template DNA is incompletely linearized. •The reaction mixture can be scaled up or down. • If the reaction product yield is decreased, 20 mM fresh DTT can be added to the reaction system.
• Transcribed fragments are less than or equal to 500bp, and it is recommended to extend the transcription time to 4-8 h.
•Precipitates are easy to be found in 10×HH T7.
