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Hotstar Taq DNA Polymerase
 

Cat#
Size,Concentration
Supplied with
PR1051
500U(2.5U/µl)
1.25ml,10℅Hotstar Taq Buffer
(Mg2+ Plus)

Store at 每20∼C
Description
Hot Start Taq DNA Polymerase is a recombinant Taq DNA polymerase which has been chemically modified by the addition of heat-labile blocking groups to its amino acid residues. The enzyme is inactive at room temperature, avoiding extension of non-specifically annealed primers or primer dimers and providing higher specificity of DNA amplification. The functional activity of the enzyme is restored during a short 4-minute incubation at 95∼C. The activated enzyme maintains the same functionality as Taq DNA polymerase: catalyzes 5'=>3' synthesis of DNA, has no detectable 3'=>5' proofreading exonuclease activity.The High Taq adds a single 3'-A overhang to each end of the PCR product, which can be applied to T/A cloning.
Features:
High yield amplification of complex templates
Short 4 min activation time
High PCR specificity, reduce effects of mispriming and primer-dimer formation
Enhanced PCR sensitivity, amplification of fragment up to 5kb
Convenient room temperature PCR set-up
Generates PCR products with 3*-A overhangs
Unit Definition:
One unit of the enzyme catalyzes the incorporation of 10nmole of deoxyribonucleotides into a polynucleotide fraction in 30min at 74⊥.
Storage Buffer:
20mM Tris-HCl, pH 8.0, 100mM KCl, 0.1mM EDTA, 5mM DTT, 50% glycerol, 0.5% NP40 and 0.5% Tween 20.
10X Reaction Buffer(with MgCl2):
200mM KCl, 100mM Tris-HCl (pH 9.0 at 25⊥) and 1% Triton X-100, 50mM (NH4)2SO4, and 15mM MgCl2.
Quality Control
No contaminating endonuclease or exonuclease activity detected.
Functionally tested in PCR.
Applications:
Hot-start PCR amplification
Specific amplification of complex cDNA and genomic template
Amplification from low copy number DNA template
Real-Time PCR
Multiple PCR
Generation of PCR products for TA cloning
Standard Protocol
1. In a sterile, nuclease-free PCR tube, combine the following:

Components
Volume
Final Concentration
10℅Hotstar Taq Buffer (Mg2+ Plus)
dNTP Mix (10 mM each)
upstream primer
downstream primer
Hotstar Taq DNA Polymerase (2.5 U/米l)
Template DNA
5 米l
1 米l
-
-
0.2-1 米l
 
variable
1 ℅
0.2 mM each
0.1每1.0米M
0.1每1.0米M
0.5-2.5 U
 
<0.5米g/50米l
Nuclease-Free Water
to 50 米l
 

2. If using a thermal cycler without a heated lid, overlay the reaction mix with 1每2 drops(approximately 50米l) of mineral oil to prevent evaporation during thermal cycling.Centrifuge the reactions in a microcentrifuge for 5 seconds.
3. Perform PCR using your standard parameters.
Recommended Thermal Cycling Conditions for Hotstar Taq DNA
Polymerase-Mediated PCR Amplification.

Step
Temperature
Time
Initial Danaturation
94∼C
3 minutes
Denaturation
94⊥
0.5-1 minutes
Annealing
42每65∼C*
0.5-1 minutes
tension
72⊥
1min/kb
Final Extension
72⊥
5 minutes
Soak
4∼C
Indefinite

4. Analyze the amplification products by agarose gel electrophoresis and visualize by ethidium bromide staining. Use appropriate molecular weight standards.
 
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