Liquid real-time PCR master mix with Sybr®Green for lyophilization of qPCR assays
2.5 x conc. master mix
| Cat. No. | Amount | Price (EUR) | Buy / Note |
|---|---|---|---|
| PCR-189-1ML | 1 ml | 143,15 | Add to Basket/Quote Add to Notepad |
| PCR-189-10ML | 10 ml | 715,77 | Add to Basket/Quote Add to Notepad |
| PCR-189-100ML | 100 ml | Ask for Quotation |
For general laboratory use.
Shipping: shipped on gel packs
Storage Conditions: store at -20 °C, avoid freeze/thaw cycles
store dark
Shelf Life: 6 months
Concentration: 2.5 x conc.
Spectroscopic Properties: SYBR®Green DNA intercalator dye bound to DNA: λexc 494 nm λem 521 nm
Description:
Liquid qPCR SybrMaster for Lyophilization is a liquid 2.5 x conc. master mix designed for custom-specific production of freeze-dried real-time PCR assays. The mix includes SYBR®Green as DNA intercalator dye and is recommended for routine PCR applications, high throughput PCR or genotyping and provides an improved specificity and sensitivity when amplifying low-copy-number targets or working with complex backgrounds.
The master mix contains all reagents required for qPCR (except template and primer) in a well balanced composition. The high specificity and sensitivity of the mix is based on a hot-start polymerase with blocked activity at ambient temperature.
The mix can also be used in combination with ROX reference dye (#PCR-356) in PCR instruments that are compatible with the evaluation of the ROX signal.
Content:
Liquid qPCR SybrMaster for Lyophilization
2.5 x conc. master mix containing antibody-blocked hot start polymerase, nucleotides, reaction buffer, SYBR®Green DNA intercalator dye, additives and stabilizers
Preparation of the 2 x conc. master mix
Liquid qPCR SybrMaster for Lyophilization comes as 2.5 x conc. master mix and must be diluted to 2.0 x concentration prior to lyophilization. Lyophilization of 10 μl, 2 x conc. master mix per tube or well to obtain a final assay volume of 20 μl is recommended.
Recommended primer and probe concentrations:
| Comp. | final conc. |
| Liquid qPCR SybrMaster for Lyophilization | 2 x conc. |
| forward Primer 1) | 300 nM |
| reverse Primer 1) | 300 nM |
| ROX Reference Dye (PCR-356) 2) | 500 nM |
| PCR-grade Water (PCR-258) | fill up to 2 x conc. |
1) The optimal concentration of each primer may vary from 100 to 500 nM.
2) Optional if ROX Refernce Dye is required for the assay design and compatible with the used cycler. 500 nM is recommended for high ROX assays.
Dispensing the master mix
Vortex the 2 x conc. mix prepared above thoroughly to assure homogeneity. Dispense 10 μl to each PCR tube or well of the plate.
Lyophilization of the master mix
Use a freeze dryer or sublimator for freeze drying the prepared master mix. Follow the instructions provided by the freeze-dryer manufacturer.
Addition of template DNA
To obtain a final assay volume of 20 μl, rehydrate the lyophilisate cakes in 20 μl template DNA / purified DNA or 20 μl PCR-grade Water for no-template controls and cap or seal the tube / plate. Do not exceed 10 ng DNA per reaction as final concentration. Tubes or plates should be centrifuged before cycling to remove bubbles.
Recommended cycling conditions:
| Initial denaturation and polymerase activation | 95 °C | 2 min | 1 x |
| Denaturation | 95 °C | 15 sec | 35-45 x |
| Annealing | 60-65 °C 3) | 15 sec | 35-45 x |
| Elongation | 72 °C | 30 sec 4) | 35-45 x |
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 and primer pair.
