» Sign in / Register

Hot Start Core Kit Ab+

Kit of antibody-blocked hot start DNA polymerase, dNTPs and reaction buffer

Cat. No. Amount Price (EUR) Buy / Note
PCR-216S 200 units 91,48 Add to Basket/Quote Add to Notepad
PCR-216L 1.000 units 365,90 Add to Basket/Quote Add to Notepad
PCR-216XL 5.000 units 1.400,26 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

Description:
Hot Start Core Kit Ab+ contains all reagents required for PCR (except template and primer) in one box combining simple handling with high flexibility.
Hot Start Polymerase Ab+ 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 initial denaturation. The thermal activation prevents the extension of nonspecifically annealed primers and primer-dimer formation at low temperatures during PCR setup. The polymerase is recommended for routine PCR applications (up to 4 kb fragment length), high throughput PCR or genotyping.
The Crystal Buffer system guarantees robust and reliable amplification results in almost all PCR applications. The buffer contains a well-balanced ratio of potassium-, ammonium- and magnesium-ions to ensure high specificity and minimal by-product formation without the need of additional optimization steps.
Ruby Buffer additionally contains gel loading buffer and an inherent red dye allowing the direct loading of the PCR product into the gel. The red dye allows an easy visual control during PCR set-up and in combination with the density reagent the direct loading of the reaction product into the gel.
The enzyme replicates DNA at 72 °C. It 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 (proof-reading) activity.

Activation step
Hot Start Polymerase Ab+ requires no prolonged heating or denaturing step. The polymerase inhibiting antibody is released within 2 min at >92°C at the initial denaturation step.

Content:
Hot Stort Polymerase Ab+ (red cap)
5 units/μl antibody-blocked hot start 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, 50 % (v/v) Glycerol, pH 8.0 (25°C)

dNTP Mix / 10 mM (white cap)
10 mM of dATP, dCTP, dGTP and dTTP

Ruby Buffer (black cap)
10 x conc. complete PCR buffer containing 200 mM Tris-HCl, KCl, (NH4)2SO4 and 20 mM MgCl2, red tracking dye and density reagent for gel loading

Crystal Buffer (green cap)
10 x conc. complete PCR buffer containing 200 mM Tris-HCl, KCl, (NH4)2SO4 and 20 mM MgCl2

componentPCR-216SPCR-216LPCR-216XL
Hot Start Polymerase Ab+200 units / 40 μl1000 units / 200 μl5000 units / 1 ml
dNTP Mix / 10 mM200 μl1 ml5 x 1 ml
Ruby Buffer1.2 ml5 x 1.2 ml25 ml
Crystal
Buffer
1.2 ml5 x 1.2 ml25 ml

Assay Set-Up:
Before starting, vortex all components thoroughly to ensure homogeneity.
Prepare a premix for the number of assays you need according to the following protocol:

comp.capstock conc.final conc.1 assay @20 μl1 assay @ 50 μl
PCR-grade Waterwhitefill up to 10 μlfill up to 30 μl
Ruby Buffer or Crystal Bufferblack or green10x1x2 μl5 μl
dNTP Mix / 10 mMwhite10 mM200 μM0.4 μl1 μl
Hot Start Polymerase Ab+red5 units/μl0.025 units/μl0.1 μl0.25 μl
primer mix or each primer10 μM each primer200 - 400 nM each primer0.4-0.8 μl1 - 2 μl
template
/sample DNA
10 μl < 10 ng DNA20 μl < 20 ng DNA

Select PCR tubes, stipes or plates as recommended for your cycler model.
Aliquot premix into each well and add template DNA (or PCR-grade Water for negative controls).

Cycling Conditions:
Spin down the tubes/plate briefly to remove bubbles and place them into the cycler.

initial
denaturation
95 °C2 min1x
denaturation
annealing1)
elongation2)
95 °C
50 - 68 °C
72 °C
10 - 20 sec
10 - 20 sec
20 sec - 4 min

25 - 35x

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.

Gel Loading and Down-Stream Applications:
Ruby Buffer (#PCR-272) includes a density reagent + tracking dye and allows the direct loading of the PCR products into a electrophoresis gel. For DNA detection / fluorescent DNA staining we recommend to use new generations dyes (i.g. SYBR DNA Stain, #PCR-273) instead of the classical but highly mutagenic ethidium bromide.
Crystal Buffer(#PCR-271) is recommended for down-stream applications such as DNA sequencing, ligation, restriction digestion or where an analysis of the PCR product by absorbance or fluorescence excitation is required. For gel electrophoresis add gel loading buffer and fluorescent DNA stain (i.g. Gel Loading Buffer with DNA Stain, #PCR-274 - #PCR-276) before loading the PCR into the gel. Using pre-stained gels or post-run staining protocols is also possible.

Additional Buffer Systems:
Labeling Buffer (#PCR-263) is recommended for DNA labeling or mutagenesis applications. The buffer is specially optimized for incorporation of labeled or modified nucleotides into DNA. It gives superior results in a broad range of reaction conditions with most primer-template pairs but amplification may also tend to an increased unspecifity.
KCl Buffer (#PCR-262) is recommended for use in routine PCR reactions. The buffer is optimized for highest specificity but may require additional fine-tuning of assay parameters like MgCl2 concentration and annealing temperature.

Optimization of MgCl2 concentration:
A final Mg2+ concentration of 2.0 mM is recommended in combination with Labeling Buffer. However, if an individual Mg2+ optimization is essential add 25 mM MgCl2 stock solution (#PCR-266) as shown in the table below.

final MgCl2 conc.20 μl final assay volume50 μl final assay volume
2 mM--
3 mM0.8 μl2.0 μl
4 mM1.6 μl4.0 μl
5 mM2.4 μl6.0 μl

Related products: