Hot Start Core Kit - red dyed

Kit of heat-activatable DNA polymerase for high specificity, dNTPs and reaction buffer, aptamer-blocked

Product Cat. No. Amount Price (EUR) Buy / Note
S pack PCR-233S-red 200 units 83,00 Add to Basket/Quote Add to Notepad
L pack PCR-233L-red 1000 units 332,00 Add to Basket/Quote Add to Notepad

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

Form: liquid

Concentration: 5 units/μl

Related products:

  • Ready-to-Use Mixes / direct gel loading
  • Ready-to-Use Mixes
  • Thermophilic Polymerases
  • Deoxynucleotides (dNTPs)
  • Supplements
  • Primers and Oligonucleotides
  • DNA Ladders

Availability Restriction: Exclusively distributed in Japan by Greiner BioOne

Description:
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.
Activation step:
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.
Kit contents:
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),
10x conc.

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 μl10x Hot Start Buffer completegreen cap
1 μldNTP Mixwhite cap
1-5 μleach Primer
(10 μM)
-
2 - 50 ngtemplate DNA-
0.2 - 0.5 μl
(1 - 2.5 units)
Hot Start Pol - redred cap
Fill up to 50 μlPCR-grade water-

Optimization of MgCl2 concentration:
A concentration of 1.5 mM Mg2+ is recommended for most applications. For an individual optimization add MgCl2 stock solution as shown in the table below.
50 μl PCR assay
MgCl2 Stock Solution1 μl2 μl3 μl5 μl
final MgCl2 conc.2 mM2.5 mM3 mM4 mM

Recommended cycling conditions:
initial
denaturation
94 °C2 min1x
denaturation94 °C30 sec30x
annealing1)45 - 68 °C30 sec30x
elongation2)72 °C30 sec - 4 min30x
final
elongation
72 °C2 min1x

1) The annealing temperature depends on the melting temperature of the primers used.
2) The elongation time depends on the length of the fragments to be amplified. A time of 1 min/kb is recommended.

For optimal specificity and amplification an individual optimization of the recommended parameters may be necessary for each new template DNA and/or primer pair.