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
Availability Restriction: Exclusively distributed in Japan by Greiner BioOne
Hot Start Core Kit 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 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 requires no prolonged heating or denaturing step. The polymerase inhibiting aptamer is quickly released at the increased temperature of thermal cycling.
|Hot Start Pol*|
10 mM each
|200 μl||1 ml|
|Hot start Buffer complete**|
|1.2 ml||5 x 1.2 ml|
|1.5 ml||2 x 1.5 ml|
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 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.