Universal protocol of quantitative PCR (qPCR) with SYBR Green
This protocol is intended for use with but not limited to: ABI PRISM®7000, 7700, and 7900HT; the ABI 5700, ABI 7300 and 7500 Real-Time PCR Systems, the Stratagene Mx3000P®, Mx3005P™, and Mx4000®, the Corbett Research Rotor-Gene™, the MJ Research DNA Engine Opticon™, Opticon® 2, and Chromo 4™ Real-Time Detector, Eppendorf Mastercycler® ep realplex, Roche LightCycler® 480, Bio-Rad CFX96
and the Cepheid Smart Cycler®.
Protocole KAPA™ SYBR® FAST qPCR Master Mix (2X) Universal
Reagents
- Template DNA
- Forward primer
- Reverse primer
- Master Mix (2X)
- PCR grade water
qPCR Protocol
Step 1: qPCR Reaction Setup
- Before preparing qPCR reactions, thoroughly mix the KAPA PROBE FAST Bio-Rad iCycler™ qPCR Master Mix (2X), template DNA, primers and probes.
-Calculate the required volumes of each component based on the following table:
| Final Concentration | 20 μl rxn | |
| PCR grade water up to 20 μl | As required | |
| qPCR Master Mix (2X) | 1X | 10 μl |
| Forward Primer (10 μM) | 200 nM | 0.4 μl |
| Reverse Primer (10 μM) | 200 nM | 0.4 μl |
| Template DNA | (<20 ng/20 μl rxn) | Variable |
| ROX High/Low (optional) | 0.4 μl |
Step 2: Plate Setup
- Transfer the appropriate volume of reaction mixture to each well of a PCR tube/plate. Reaction volumes may be scaled down from 20 μl to 10 μl if low volume tubes/plates are used.
- Cap or seal the reaction tube/plate and centrifuge briefly.
Step 3: Run the qPCR reaction
- If applicable, select fast mode on the instrument.
- Program the following cycling protocol:
- Enzyme activation at 95 °C during 20 sec - 3 min (1 cycle)
- Denature at 95 °C during 1 - 3 sec
- Anneal/Extend/Acquire at 60 ºC ≥ 20 sec
- Dissociation according to instrument guidelines Do 40 cycles of points 2 and 3.
Step 4: Analyze the results
- Data analysis varies depending on the instrument used. Please refer to your instrument user guide for information.
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