This protocol is intended for use with but not limited to: ABI PRISM™7000, 7700, 7900HT, the ABI 7300 Real-Time PCR System, the GeneAmp®5700, the StepOne™ and the StepOnePlus™ instruments. This kit is not compatible with instruments that use ROX at a final concentration lower than 500 nM.
These kits are ideally suited for: Gene expression analysis, SNP genotyping, Microarray validation and Gene knockdown validation.
- Template DNA or cDNA
- 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)
||100 - 400 nM
|Reverse Primer (10 μM)
||100 - 400 nM
||100 - 500 nM
|Template DNA or cDNA
- 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 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.
KAPA PROBE FAST qPCR Master Mix (2X) ABI Prism™ Protocol
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