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Multiplex Master Mix | Master Mixes Cellco
Multiplex Master Mix
Ficha técnica
| Código | Multiplex Master Mix |
| Categoria | PCR Convencional |
| Marca | Cellco |
Descrição Geral
Master Mix High Fidelity (2X) | Master Mixes - Cellco
Master mix com polimerase termoestável de alta fidelidade
Pronto para uso
For in vitro use only!
Envio: Shipped on blue ice
Condições de armazenamento: Store at -20 °C
Avoid freeze/thaw cycles.
Validade: 12 months
Kit contents:
2x Multiplex PCR Master (purple cap)
Master mix of thermostable Hot Start DNA polymerase, dATP, dCTP, dGTP, dTTP, KCl, MgCl2 and stabilizers.
Master mix of thermostable Hot Start DNA polymerase, dATP, dCTP, dGTP, dTTP, KCl, MgCl2 and stabilizers.
PCR grade water (white cap)
Descrição:
Multiplex PCR Master is specially designed for the set-up of multiplex PCR reactions. It contains an optimized composition of polymerase, nucleotides, MgCl2 and stabilizing components in a specifically developed buffer system allowing the parallel amplification of a multitude of fragments in a single PCR assay. The master mix contains all reagents (except primer and template) in a 2x concentrated ready-to-use solution. The kit is recommended for use in clinical PCR reactions and highly suitable for multiple target gene amplification in a single tube. The high specificity and sensitivity of the mix is achieved by a chemically inhibited hot-start polymerase. Its activity is blocked at ambient temperature preventing the extension of nonspecifically annealed primers and primer-dimer formations at low temperatures during PCR setup.
PCR Reaction Setup:
A reaction volume of 10-50 µl per assay is recommended for most PCR cyclers. Pipet with sterile filter tips and perform the setup in an area separate from DNA preparation or analysis. Notemplate controls should be included in all amplifications. Thaw, mix, and briefly centrifuge each component before use. Add the following components to a microcentrifuge tube:
1. Prepare PCR master mix
Note: Consider the volumes for all components listed next steps to determine the correct amount of water required to reach your final reaction volume.
| Component | 50 μL rxn | Final conc. |
| Water, grade PCR | to 50 μl | - |
| 2 X Taq High Fidelity Master Mix | 25 μl | 1x |
Mix and briefly centrifuge the components.
2. Add template DNA and primers
| Component | 50 μl rxn | final conc. |
| Forward primer (10 μM) | 0,5 – 2,5 μl | 0,1 – 0,5 μM |
| Reverse primer (10 μM) | 0,5 – 2,5 μl | 0,1 – 0,5 μM |
| Template DNA | x μl | 10 pg – 1 μg* |
*genomic DNA: 1 ng – 1 μg; plasmidial DNA: 1 pg – 1 ng
Cap each tube, mix, and briefly centrifuge the content.
3. Incubate reactions in a thermal cycler
Recommended cycling conditions:
| Initial denaturation | 95 °C | 10 min | 1x |
| Denaturation | 95 °C | 15 - 30 sec | 30 - 50 cycles |
| Annealing** | 58 - 64 °C | 15 - 40 sec | 30 - 50 cycles |
| Elongation*** | 72 °C | 1 min/kb | 30 - 50 cycles |
| Final extension (optional) | 72 °C | 5 min | 1x |
| Hold | 4 – 8 °C | ∞ | 1x |
**The optimal annealing temperature (AT) can be calculated for each primer as following: AT = Tm - 5 °C with Tm = 2 °C x (A+T) + 4 °C x (G+C) Please note that primers should be designed to show minimal differences in there melting temperatures (Tm). 2)The elongation time d
***The elongation time depends on the length of the fragments to be amplified. A time of 1 min/kb is recommended.
For optimal specificity and amplification an individual optimization of the recommended parameters may be necessary for each new template DNA and/or primer pair.
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