qPCR GreenMaster Lyophilisate

For general laboratory use.

Shipping: shipped at ambient temperature

Storage Conditions: store at ambient temperature
Store in an aluminium-coated bag or on a dry place.
Lyophilisates may hydrate at humidity levels >70 % when sealing is opened.

Shelf Life: 12 months in sealed package

Description:
qPCR GreenMaster Lyophilisate is designed for quantitative real-time analysis of DNA samples using a green-fluorescent DNA stain structurally similar to SYBR® GREEN. The fluorescent dye in the master mix intercalates into the amplification product during the PCR process and enables the rapid analysis of target DNA without the need to synthesize sequence-specific labeled probes. It provides an easy-to-handle and 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 lyophilisate contains all reagents required for qPCR (except template, primer and labeled fluorescent probe) in a single bead. The high specificity and sensitivity of the mix based on 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.

Content:
qPCR GreenMaster Lyophilisate
antibody-blocked hot start polymerase, dATP, dCTP, dGTP, dTTP, KCl, (NH₄)₂SO₄, MgCl₂, green-fluorescent DNA stain, additives and stabilizers

PCR-grade water

Handling
The lyophilisates are provided in low-profile (0.1 ml) 96-well plates with optically clear caps, whereby each well contains reaction mix for a final volume of 20 μl. The plates can be easily divided into 8-well strips and further segmented by cutting, allowing compatibility with a variety of PCR cyclers.
The lyophilisate combines highest performance with convenience of use and stability. There is no need for freezing, thawing or pipetting on ice. The few remaining pipetting steps minimize the risk of errors or contaminations.
Each vial contains all components (except primers and template) required for a 20 μl real-time PCR assay.
To perform PCR, only fill up the vials with a primer mix and add DNA template.
The lyophilisate 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 (#PCR-351) should be added as 1x concentration to the PCR reaction.

Recommended PCR assay:

¹⁾ The optimal concentration of each primer may vary from 100 to 500 nM.

Preparation of the primer mix
Prepare 13 volumes of primer mix for 12 samples or a triple-set of 4 samples as specified. 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 (NTC) should be included in all applications.

Dispensing the master mix
Vortex the primer/probe mix thoroughly to assure homogeneity. Dispense 15 μl to each PCR tube or well of the plate.

Addition of template DNA
Add 5 μl of template DNA (or no-template controls) to each reaction vessel and cap or seal the tube / plate. Do not exceed 200 ng DNA per reaction as final concentration. Tubes or plates should be centrifuged before cycling to remove possible bubbles.

Recommended cycling conditions:

²⁾ The annealing temperature depends on the melting temperature of the primers used.
³⁾ The elongation time depends on the length of the amplicon. A time of 30 sec for a fragment of up to 500 bp is recommended.

For optimal specificity and amplification an individual optimization of the recommended parameters, especially of the annealing temperature may be necessary for each new combination of template DNA and primer pair.

® SYBR is a trademark of Molecular Probes, Inc.

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