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T7 RNA Polymerase HCP1011A Featured Image
  • T7 RNA Polymerase HCP1011A

T7 RNA Polymerase


Cat No.:HCP1011A

Package: 100μL/1mL/10mL/100mL

T7 RNA Polymerase is a DNA-dependent RNA polymerase from T7 phage

Product Description

Procduct data

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.


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  • 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.

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