qPCR Protocol with SYBR Green for ABI Prism™

This protocol is intended for use with most ABI qPCR cyclers.

Protocole KAPA™ SYBR® FAST qPCR Kit Master Mix (2X) ABI Prism™




Reagents
- Template DNA
- 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	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

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
Make 40 cycles for the last 2 points.

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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