qPCR Protocol with Taqman® probe
This protocol is intended for use with, but not limited to: the 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 Rotor-Gene Q, the MJ Research DNA Engine Opticon™, Opticon® 2 and Chromo 4™ Real-Time Detector, the Eppendorf Mastercycler® ep realplex, the Roche LightCycler® 480, the Bio-Rad CFX96 and the Cepheid Smart Cycler®.
These kits are ideally suited for: Gene expression analysis, SNP genotyping, Microarray validation and Gene knockdown validation.Reagents
- Template DNA or cDNA
- Forward primer
- Reverse primer
- Probe
- 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 | 20μl rxn | |
| PCR grade water up to 20μl | As required | |
| qPCR Master Mix (2X) | 1X | 10μl |
| Forward Primer (10μM) | 100 - 400 nM | Variable |
| Reverse Primer (10μM) | 100 - 400 nM | Variable |
| Probe | 100 - 500 nM | Variable |
| Template DNA or cDNA | <250 ng | Variable |
Step 2: Plate Setup
- Preparation of a reaction cocktail is vital in qPCR to reduce the effect of pipetting errors between samples. Assemble all components above except template DNA or cDNA.
- Gently mix all components in the cocktail before transferring the appropriate volume of reaction mixture to each well of a PCR tube/plate.
- Add template DNA or cDNA to each reaction.
- Reaction volumes may be scaled down from 20μl to 10μl 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 Make 40 cycles for the last 2 points.
- Data analysis varies depending on the instrument used. Please refer to your instrument user guide for information.
Product list
KAPA™ PROBE FAST qPCR Kit Master Mix (2X) Universal Protocol