T7RNA polymerase is a DNA-dependent RNA polymerase derived from T7 phage with highly specific 5'→3' terminal RNA polymerase activity. T7RNA polymerase has high specificity to the T7 promoter and can synthesize a large amount of RNA using the downstream sequence of the T7 promoter as a template.
Product Content
| T292917 | Component | 50 μL | 250 μL | 1 mL | Storage | | T292917A | T7 RNA Polymerase | 50 μL | 250 μL | 1 mL | -20°C. Avoid freeze/thaw cycle | | T292917B | 10×HH T7 Buffer | 2×50 μL | 2×250 μL | 2×1 mL | -20°C. Avoid freeze/thaw cycle |
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Activity unit definition
In a standard reaction system, the amount of enzyme required to catalyze NTP to produce a 1nmol PPi within 37°C1 hours is defined as one unit of activity (U).
Iinstructions
System configuration (50 μ l）
| 组分 | 体积 |
Nuclease-free water/DEPC
| Up to 20 μL
|
10×T7 RNA Polymerase Buffer
| 2 μL
|
ATP/GTP/CTP/UTP (100mM each)
| 0.4 μL each (2mM each Final)
|
RNase inhibitor
| 1 μL (40 U)
|
Inorganic pyrophosphatase
| 0.5 μL (0.05 U)
|
T7 RNA Polymerase (50 U/μL)
| 1 μL
|
Linearized template DNA
| 1 μg
|
10×HH T7 Buffer: 400 mM Tris-HCl (25℃，pH 8.20), 60 mM MgCl2, 100mM DTT, 20mM spermidine.
The addition of reaction components *10xHH T7 Buffer in the above order is only applicable to the dosage of NTPS with a final concentration of 2mM. If the dosage of 7.5mM to 10mM is required, please choose the high-yield kit series.
Reaction time: Incubation at 37℃ for 2 h.
Reaction termination: Add 2μL 0.2M EDTA (pH = 8.0@25 °C) or heat at 75° C for 10 min.
DNA template removal: DNasel(2 U) at 37°C for 15 min.
Quality Control
1) Endonucrenase residue detection: 50U benzyme and 1μg λDNA were added to the 50μL reaction system at 37°C for 16 hours, and the DNA bands of agarose gel electrophoresis did not change.
2) Exonuclide residue detection: 50U benzyme and 1μgλ-HindIIIdigestDNA were added to the 50μL reaction system and incubated at 37°C for 16 hours, and the DNA bands of agarose gel electrophoresis did not change.
3) Nucleic acid incision enzyme residue detection: 50U benzyme and 1μgpBR322DNA were added to the 50μL reaction system and incubated at 37°C for 16 hours, and the DNA bands of agarose gel electrophoresis did not change.
4) RNase residue detection: 50U benzyme and 1.6μgMS2RNA were added to the 50μL reaction system and incubated at 37°C for 16 hours, and the RNA bands of agarose gel electrophoresis did not change.
5) Thermal inactivation: 75°C, 10min.
Matters needing attention
1. The preparation of transcription reaction should be completed in an environment free of RNase pollution, and gloves are recommended during operation. The system was prepared using nuclease-free water, suction heads and reaction tubes.
2, you can increase the RNA production by increasing the NTP concentration (each concentration can reach 10mM), while the need to appropriately increase the Mg "concentration.
3, the reaction system needs to be prepared at room temperature to avoid DNA reaction with 10xHH T7 Buffer at 4 ℃ and precipitate.
4. Incomplete linearization of template DNA may reduce the yield and length of transcription products.
5, the volume of the reaction system can be scaled up or down according to actual needs.
6. If the reaction product decreases, 20 mM fresh DTT can be added to the reaction system.
7, transcription fragments ≤500bp, it is recommended to extend the transcription time to 4-8 h.
8. 10xHH T7 Buffer is prone to precipitate after freezing. Please confirm whether it is completely dissolved before use. If precipitate occurs, it can be shaken and redissolved after fully heating at 37 ℃
9, 10xHH T7 Buffer is only suitable for 2mM final concentration of NTPS feed, such as 7.5mM-10mM feed, please choose high yield kit series products.
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