Features:
- The PCR-Master mix contains dUTP instead of dTTP
- The MasterMix for PCR contains UNG (Uracil-N-Glycosylase)
- The PCR-Mix contains ROX as
passive Reference dye (it provides a baseline in multiplex reactions)
- It contains EvaGreen as fluorescent dye
- The qPCR / RTD-PCR Master mix E4 is ready-to-use and is optimized
for high specificity and sensitivity because of optimized reaction buffer
- easy to use because ready-to-use Master Mix
Applications:
- Detection and quantification of DNA and cDNA targets
- Profiling gene expression
- Microbial detection
- Viral load determination
Compatibility:
ABI: 5700, 7000, 7300, 7700, 7900, 7900HT, 7900HT Fast, StepOne, StepOne plus
Description:
The PCR-Master Mix contains all reagents required for qPCR (except template and primer) in a premixed 2x concentrated ready-to-use solution. The high
specificity and sensitivity of the mix 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 non-specifically annealed primers and primer-dimer formations at low temperatures during PCR setup.
The PCR-mix offer dUTP instead of dTTP to prevent carry-over contaminations of DNA from previous PCR reactions.
Concentration: The Mastermix is 2x concentrated
List of components qPCR / RTD-PCR Master mix:
Hot-Start Polymerase (anti-body blocked) for qPCR, dATP, dCTP, dGTP, dUTP, EvaGreen, High-ROX, UNG, optimized reaction buffer
with KCl and MgCl2, stabilizers and enhancers, PCR-grade water
Transportation: with blue ice
Storage: at 4°C for 3 months, at -20°C for more than 12 months,
Note: protect from Light
Manufactured and quality-controlled in accordance with ISO 9001 and 14001
Components | Volume per reaction | final conc. |
2X qPCR / RTD- PCR Master mix E4 |
25 µl | 1x |
Up-stream primer (10 µM stock) |
1,5 µl (range: 0,5-2.5 µl) | 300 nM |
Down-stream primer (10µM stock) |
1,5 µl (range: 0.5-2,5 µl | 300 nM |
Template DNA |
5 µl (0.1-15 ng/ml plasmid DNA) (1-10 µg/ml genomic DNA) |
< 500ng DNA |
Sterile dest. Water (included) |
up to 50 µl total reaction volume |
- vortex all solutions carefully before using and before PCR
- may you add the enzyme mix after Template DNA
- an individual optimization of annealing temperature may be
necessary for new combinations of primers and Template DNA
General Thermo-Cycler protocol for qPCR / RTD-PCR Master mix:
Note: working with EvaGreen just select the optical setting for FAM or SYBR Green at the cycler
Step | Time | Temperature |
Initial denaturation | 1-3 min | 95°C |
30-40 Cycles: Denaturation Annealing Extension |
15-30 sec 30-65 sec 30 sec (per 500bp) |
95°C 55-65°C 72-75°C |
Note:
an individual optimization of annealing temperature may be necessary for new combinations of primers and Template DNA
PCR MASTERMIX SELECTION TABLE
PCR Mastermixes with dTTP and SybrGreen
BioStar 2 PCR mastermix with SYBRGreen, blue dye, no
rox
BioStar 3 PCR mastermix with SYBRGreen, 60 nm LOW-ROX
BioStar 4 PCR mastermix with SYBRGreen, 900 nm HIGH-ROX
PCR Mastermixes with dTTP for probes
BioStar 5 PCR mastermix for probes
BioStar 6 PCR mastermix for probes 60 mM LOW-ROX
BioStar 7 PCR mastermix for probes 900 mM HIGH-ROX
qPCR Mastermix E4