Kit of heat-activatable DNA polymerase for high specificity, dNTPs and reaction buffer, aptamer-blocked
For in vitro use only!
Unit Definition: One unit is defined as the amount of the enzyme required to catalyze the incorporation of 10 nmoles of dNTP's into an acid-insoluble form in 30 minutes at 70 °C using hering sperm DNA as substrate.
Shipping: shipped on blue ice
Storage Conditions: store at -20 °C
avoid freeze/thaw cycles
Shelf Life: 12 months
Concentration: 5 units/μl
Hot Start Core Kit - red contains all reagents required for PCR (except template and primer) in one box combining simple handling with high flexibility. The remium quality polymerase, ultrapure dNTPs and the optimized complete reaction buffer ensure superior amplification results. The additional reaction buffer without MgCl2 in combination with the MgCl2 stock solution allows an easy optimization of difficult amplifications. The kit contains Hot Start Pol - red for easier handling and to facilitate the preparation of the master mix.
The kit provides improved specificity and sensitivity when amplifying low-copy-number targets in complex backgrounds or when prolonged room-temperature set up is required. The polymerase activity is blocked at ambient temperature and switched on automatically at the onset of the initial denaturation. The thermal activation prevents the extension of nonspecifically annealed primers and primer-dimer formation at low temperatures during PCR setup.
The enzyme catalyzes the polymerization of nucleotides into duplex DNA in 5'→3' direction in the presence of magnesium. It also possesses a 5'→3' polymerization-dependent exonuclease replacement activity but lacks a 3'→5' exonuclease activity.
Hot Start Pol - red requires no prolonged heating or denaturing step. The polymerase inhibiting aptamer is quickly released at the increased temperature of thermal cycling.
Hot Start Core Kit - red contains Hot Start Pol - red for easier handling and to facilitate the preparation of the master mix.
Hot Start Pol - red (red cap)
5 units/μl Taq DNA Polymerase in 20 mM Tris-HCl, 100 mM KCl,
0.1 mM EDTA, 1 mM DTT, 0.5 % Tween-20, 0.5 % Nonidet P-40, inert red dye, 50 % (v/v) Glycerol, pH 8.0 (25°C)
dNTP Mix (white cap)
10 mM each dNTP (dATP, dCTP, dGTP, dTTP)
Hot Start Buffer complete (green cap),
200 mM Tris-HCl, 500 mM KCl, 15 mM MgCl2, pH 8.5 (25°C)
MgCl2 Stock Solution (yellow cap)
25 mM MgCl2
Recommended 50 μl PCR assay:
|5 μl||10x Hot Start Buffer complete||green cap|
|1 μl||dNTP Mix||white cap|
|1-5 μl||each Primer|
|2 - 50 ng||template DNA||-|
|0.2 - 0.5 μl|
(1 - 2.5 units)
|Hot Start Pol - red||red cap|
|Fill up to 50 μl||PCR-grade water||-|
|MgCl2 Stock Solution||1 μl||2 μl||3 μl||5 μl|
|final MgCl2 conc.||2 mM||2.5 mM||3 mM||4 mM|
|94 °C||2 min||1x|
|denaturation||94 °C||30 sec||30x|
|annealing1)||45 - 68 °C||30 sec||30x|
|elongation2)||72 °C||30 sec - 4 min||30x|
|72 °C||2 min||1x|
For optimal specificity and amplification an individual optimization of the recommended parameters may be necessary for each new template DNA and/or primer pair.
Availability Restriction: Exclusively distributed in Japan by Greiner BioOne