qPCR

MasterMixes

Hot Start PCR allowing to achieve highest sensitivity and specificity under both standard and fast qPCR cycling conditions.
ROX content option for best normalization calculations in different concentration to fit for almost all type of instruments.
Works with high GC & high AT-rich templates
Rapid extension, early Ct detections
Very high sensitivity
Minimum or no optimization is required

Relative gene expression analysis
Absolute quantification of any RNA template (mRNA, total RNA, viral RNA) and low copy number genes
qRT-PCR assays based on CYBER Green or specific Probes: including TaqMan®, Molecular Beacons, Scorpions™ Probes

2X master mix for 500 or 1000 reactions per 20µl reaction volume
Supplied with PCR degree water
To cover all RT-PCR instruments, the InView qRT-PCR MasterMixes are available in three versions – without ROX, with low or high ROX concentration. See the ROX concentration table below to know which ROX concentration suits your instrument

Tips for successful workflow

Use special primer selection programs for good planning.
Work with amplicons in a range of 80-200, max 400 bp.
Take typical measures to prevent PCR cross over contamination, keep your bench clean, wear gloves, use sterile tubes and filter pipet tips.
Run reactions in triplets; include a no-template control, no RT Mix control and positive control in parallel.
Thaw and keep reagents on ice. Mix well before use.
Do not perform annealing/extension for more than 30 seconds and do not use lower than 60 °C temperature for this step.

To Prepare a 20 μl reaction, mix the following:

Reverse Primer: 100 - 400 nM final concentration
Forward Primer: 100 - 400 nM final concentration
Specific Probe 200 nM final c. (0.4 μl of 10 μM) "Only if Probe based MasterMix is used. CYBER Green MasterMixes do not require Probes"
cDNA template 100 ng or gDNA 1µg
Add PCR Water to 10 μl volume
InView qRT-PCR MasterMix (2X): 10 μl
Mix gently, avoid bubbles. Place into the instrument
- If Probe based MasterMixe is used set the instrument channels according to the probes specifications
- If CYBER Green MasterMix is used, set the instrument onto SYBR® Green or FAM channel

PCR Program

Initial denaturation 1 cycle: 95°C - 2 min for cDNA, or 1 cycle: 95°C - 3 min for gDNA
Denaturation 40 cycles: 95°C - 5 sec
Annealing/extension 40 cycles: 60-65°C – 20-30 sec

rox2