This protocol is intended for use with most ABI qPCR cyclers.
Protocole KAPA™ SYBR® FAST qPCR Kit Master Mix (2X) ABI Prism™
- Template DNA
- Forward primer
- Reverse primer
- Master Mix (2X)
- PCR grade water
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:
Step 2: Plate Setup
||20 μl rxn
|PCR grade water up to 20 μl
|qPCR Master Mix (2X)
|Forward Primer (10 μM)
|Reverse Primer (10 μM)
||(<20 ng/20 μl rxn)
- 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:
Step 4: Analyze the results
- 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.
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