Kit for quantitative real-time PCR using labeled DNA probes
For in vitro use only!
Shipping: shipped on blue ice
Storage Conditions: store at -20 °C
avoid freeze/thaw cycles
Shelf Life: 12 months
qPCR Probes Core Kit is designed for the quantitative real-time analysis of DNA samples using DNA probe based detection. The kit is recommended for use with Dual-Labeled Fluorescent Probes, e.g. TaqMan®, Molecular Beacons or FRET probes. It provides a powerful tool for quantification of sample DNA in a broad dynamic range of up to 6 orders of magnitude with exceptional sensitivity and precision.
The high specificity and sensitivity of the Core Kit is achieved by an optimized hot-start polymerase. Its 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 included dNTP mix contains dUTP instead of dTTP and allows an UNG (Uracil-N-Glycosylase) treatment at the onset of thermal cycling to prevent carry-over contaminations of DNA from previous PCR reactions.
The reaction chemistry of the kit is optimized for block-based PCR instruments. The kit can also be used with ROX reference dye in PCR instruments that are compatible with the evaluation of the ROX signal. In this case, the ROX dye should be added as 1x concentration to the PCR reaction.
qPCR Pol (red cap)
dNTP Mix incl. dUTP (white cap)
10 mM dATP, 10 mM dCTP, 10 mM dGTP, 20 mM dUTP
qPCR ProbesBuffer complete - 10x conc. (green cap)
200 mM Tris-HCl (pH 8.5), KCl, (NH4)2SO4, 30 mM MgCl2 and stabilizers
qPCR ProbesBuffer without MgCl2 - 10x conc. (blue cap)
200 mM Tris-HCl (pH 8.5), KCl, (NH4)2SO4 and stabilizers
MgCl2 Stock Solution (yellow cap)
25 mM MgCl2
|30 μl||130 μl|
|dNTP Mix incl. dUTP|
|120 μl||600 μl|
|qPCR ProbesBuffer complete - 10x conc.|
|1 ml||3 x 1 ml|
|qPCR ProbesBuffer without MgCl2 - 10x conc.|
|1 ml||3 x 1 ml|
|MgCl2 Stock Solution|
|1.2 ml||5 x 1.2 ml|
Dual-labeled DNA probes:
Real-time PCR technology based on dual-labeled DNA probes provides a high sensitive and high specific PCR system with multiplexing capability. It requires two standard PCR primers and the DNA probe that hybridizes to an internal part of the amplicon. The sequence of the dual-labeled DNA probe should avoid secondary structure and primer-dimer formation.
Preparation of the qPCR master mix:
The preparation of a master mix is crucial in quantitative PCR reactions to reduce pipetting errors. Prepare a master mix of all components except template as specified. A reaction volume of 20-50 μl is recommended for most real-time instruments. Prepare 13 volumes of master mix for 12 samples or a triple-set of 4 samples. Pipet with sterile filter tips and minimize the exposure of the labeled DNA probe to light. Perform the setup in an area separate from DNA preparation or analysis. No-template controls should be included in all amplifications.
|component||1x 50 μl|
|13x 50 μl|
|qPCR ProbesBuffer complete||5 μl||65 μl||1x|
|dNTP Mix incl. dUTP||1 μl||13 μl||200 μM|
|1.5 μl||19.5 μl||300 nM|
|1.5 μl||19.5 μl||300 nM|
|1 μl||13 μl||200 nM|
|UNG (PCR-353)3)||0.2 μl||2.6 μl||0.2 units/assay|
|qPCR Pol||0.25 μl||3.25 μl||0.025 units/μl|
|PCR-grade water||fill up to|
|fill up to|
Optimization of MgCl2 concentration:
A concentration of 3.0 mM Mg2+ as provided by the 10x qPCR Buffer complete (green cap) is recommended for most applications. For an individual optimization use the reaction buffer without MgCl2 and add MgCl2 Stock Solution as shown in the table below.
50 μl assay
|MgCl2 Stock Sol.||4 μl||6 μl||8 μl||12 μl|
|final MgCl2 conc.||2 mM||3 mM||4 mM||6 mM|
Addition of template DNA:
Add 10 μl of sample template DNA to each reaction vessel containing 40 μl master mix and cap or seal the tube / plate. Do not exceed 500 ng DNA per 50 μl reaction as final concentration. Tubes or plates should be centrifuged before cycling to remove possible bubbles.
Recommended cycling conditions:
|UNG treatment4)||50 °C||2 min||1x|
|95 °C||2 min||1x|
|Denaturation||95 °C||15 sec||30-40x|
|60-65 °C5)||30 sec6)||30-40x|
For optimal specificity and amplification an individual optimization of the recommended parameters, especially of the MgCl2 concentration and the annealing temperature may be necessary for each new combination of template DNA, primer pair, and DNA probe.