qPCR GoldMaster
Master mix para real-time PCR com corante de DNA EvaGreen e GoldTaq hot start polimerase quimicamente modificada
| Catálogo Nº | Apresentação | Preço (R$) | Comprar |
|---|---|---|---|
| PCK-200XS | 1 x 1,25 ml | 214,16 | Adicionar ao Carrinho |
| PCK-200S | 2 x 1,25 ml | 428,32 | Adicionar ao Carrinho |
| PCK-200M | 4 x 1,25 ml | 856,64 | Adicionar ao Carrinho |
| PCK-200L | 10 x 1,25 ml | 2.034,11 | Adicionar ao Carrinho |
| PCK-200XL | 20 x 1,25 ml | 3.660,30 | Adicionar ao Carrinho |
| PCK-200XXL | 40 x 1,25 ml | 6.954,57 | Adicionar ao Carrinho |
For in vitro use only!
Envio: shipped on blue ice
Condições de armazenamento: store at -20 °C
avoid freeze/thaw cycles, store dark
Validade: 12 months
Forma: liquid
Concentração: 2x conc.
Contente:
qPCR Gold Master (red cap)
chemically-blocked GoldTaq DNA polymerase hot start, dATP, dCTP, dGTP, dTTP, KCl, reaction buffer with (NH4 2SO4, MgCl2, EvaGreen® DNA intercalator dye, additives and stabilizers
EvaGreen® Fluorescent DNA Stain:
EvaGreen® Fluorescent DNA Stain is a superior DNA intercalator dye specially developed for DNA analysis applications including real-time PCR (qPCR) and high-resolution DNA melting curve analysis (HRM). Upon binding to DNA, the non-fluorescent dye becomes highly fluorescent while showing no detectable inhibition to the PCR process. The dye is extremely stable both
thermally and hydrolytically, providing convenience during routine handling. The high quantum yield, excellent stability and lowest inhibition toward PCR makes it the ideal fluorophore in real-time PCR applications and a superior replacement for the widely used SYBR® Green I dye.
To perform the EvaGreen-based assay simply select the optical setting for SYBR® Green or FAM on the detection instrument.
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 master mix to light. Perform the setup in an area separate from DNA preparation or analysis. No-template controls should be included in all amplifications.
| component | 20 μl assay | 50 μl assay | final conc. |
| qPCR Gold Master | 10 μl | 25 μl | 1x |
| primer forward (10 μM)1) | 0.6 μl | 1.5 μl | 300 nM |
| primer reverse (10 μM)1) | 0.6 μl | 1.5 μl | 300 nM |
| UNG2) | 0,2 μl | 0,2 μl | 0,2 U/assay |
| template DNA | x μl | x μl | <500 ng |
| PCR-grade water | fill up to 20 μl | fill up to 50 μl | - |
1) The optimal concentration of each primer may vary from 100 to 500 nM.
2) Only required if an UNG (Uracil-N-Glycosylase) treatment to prevent carry-over contaminations of DNA should be applied. UNG is not provided by this kit.
Dispensing the master mix:
Vortex the master mix thoroughly to assure homogeneity and dispense the mix into real-time PCR tubes or wells of the PCR plate.
Addition of template DNA:
Add the remaining x μl of sample/template DNA to each reaction vessel containing the master mix and cap or seal the tubes/plate. Do not exceed 500 ng DNA per reaction as final concentration. Tubes or plates should be centrifuged before cycling to remove possible bubbles.
Recommended cycling conditions:
| UNG treatment3) | 50 °C | 2 min | 1x |
| Initial denaturation and polymerase activation | 95 °C | 2 min | 1x |
| Denaturation | 95 °C | 15 sec | 50 cycles |
| Annealing | 60-65 °C4) | 15-60 sec | 50 cycles |
| Elongation | 72 °C | 1 min/kb | 50 cycles |
3) Cycling step 1 is only required if an UNG (Uracil-N-Glycosylase) treatment is applied
4) The annealing temperature depends on the melting temperature of used primers.
5) The elongation time depends on the length of the amplicon. A time of 30 - 60 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.
EvaGreen® is a registered trademark and licensed for sale by Biotium, Inc., Hayward, CA, USA.