m-Superhot Taq DNA Polymerase for real time PCR and Hot-start PCR: Datasheet
Features: Maximo M-Superhot Taq DNA Polymerase for qPCR is designed for Real-Time PCR and Hot-start PCR. A special inhibitor suppresses the reaction at room temperature until after the first denaturation step. This prevents primer-dimers and other artefacts. Using the enzyme there is no need to adjust the existing standard PCR protocol.
Applications:
- Hot Start and real time PCR
- Multiplex PCR
- Amplification of complex genomic and cDNA templates
- no primer-dimers and other artêfacts; inactive at room temperature
- short activation time for real time PCR
- enhanced PCR sensitivity
Description:
Maximo M-Superhot Taq DNA for qPCR and Hot-Start-PCR is an optimized mixture of a high processive Taq DNA Polymerase and special inhibitors to Taq DNA for real time PCR. The enzyme is a thermostable DNA polymerase that possesses a 5´→3´ polymerase activity and a double-stranded specific 5´→3´ exonuclease activity. The enzyme consists of a single polypeptide with a molecular weight of 94kDa. It is developed for real time PCR or as basis enzyme for real time PCR diagnostics systems.
Concentration: 5 u/µl
Unit definition:
One unit is defined as the amount of enzyme that incorporates 10 nmoles of dNTP's into acid-insoluble form in 30 minutes at 74oC under assay conditions:
25mM TAPS pH 9.3 at 25oC, 50mM KCl, 2mM MgCl2; 1mM beta-mercaptoethanol; 200µM each dATP, dGTP, dTTP and 100 µM dCTP (a mix of unlabled and µ-[32P]-labled); 12.5 µg activated salmon sperm DNA in the final volume of 50 µl
Storage Buffer:
20 mM Tris-HCl (pH 8.0), 100 mM KCl, 0.1 mM EDTA; 1 mM DTT, 50 % Glycerol, 0.5 % Nonident P-40, 0.5 % Tween-20
Reaction Buffer:
Reaction buffer (10X)” incomplete” (red cap):160 mM (NH4)2SO4, 670mM TrisHCl pH8,8, 0,1% Tween-20
Reaction buffer (10X) “complete” (yellow cap):160 mM (NH4)2SO4, 670mM TrisHCl pH8,8, 0,1% Tween-20, 25mM MgCl2
separate Tube: MgCl2 (100 mM, green cap)
Quality control:
Activity and performance test in real time PCR, SDS-PAGE purity, absence of endonucleases/nickases and exonucleases test
Usage:
Use your existing and optimized protocol. In contrast to chemically modified Taq DNA pol. where the first denaturation step needs up to 15 min, Maximo M-Superhot Taq for Real Time PCR does not need a prolonged heating or denaturation time and works excellent basis enzyme for real time PCR.
|
Components |
Volume per reaction |
|
10X reaction buffer |
10 µl |
|
100 mM MgCl2 |
optional |
|
dNTP-Mix (40mM) |
1.0 µl |
|
Up-stream primer (10 µM stock) |
0,5-2.5 µl |
|
Down-stream primer (10µM stock) |
0.5-2,5 µl |
|
Template DNA |
0.1-15 ng/ml plasmid DNA
1-10 µg/ml genomic DNA |
|
Maximo M-Superhot Taq DNA (5 u/µl) |
0.2 - 1.0 µl |
|
Sterile dest. Water (molecular grade) |
up to 50 µl total reaction volume |
Note:
- vortex all solutions carefully before using
- add the enzyme after Template DNA
- may you have to optimize the MgCl2 concentration for best result
General Thermo-Cycler protocol:
|
Step |
Time |
Temperature |
|
Initial denaturation |
2-5 min |
94-95°C |
25-30 Cycles:
Denaturation
Annealing
Extension
|
10-25 sec
10-25 sec
60 sec |
94-95°C
55-65°C
72°C per 1kb
|
|
Final extension |
5 min |
72°C |
Note:
- In case of low amount of DNA template, additionally cycles may be used
Transportation: on blue ice
Storage: at -20°C for 24 months or for more than 3 months at +4°C
|
