1-step qRT-PCR Protocol for ABI Prism™
This protocol is intended for use with the ABI PRISM® 7000, 7700, 7900HT, ABI 7300 Real-Time PCR Systems, GeneAmp® 5700, StepOne™, StepOnePlus™, and ViiA™ 7. This kit is not compatible with instruments that use ROX at a final concentration lower than 500 nM.
Protocole KAPA™ SYBR® FAST One-Step qPCR ABI Prism™
Réactifs :
- Template RNA
- Forward primer
- Reverse primer
- KAPA SYBR® FAST qPCR Master Mix (2X) with ROX Reference Dye
- dUTP
- KAPA RT Mix (50X)
Protocole général de qPCR
Step 1: qPCR Reaction Setup
- Before preparing qRT-PCR reactions, thoroughly mix the KAPA SYBR® FAST qPCR Master Mix (2X), KAPA RT Mix (50X), dUTP (10 mM), template RNA and primers. ROX reference dye is included in the Master Mix at a final concentration of 500 nM.
- Keep all kit components and assemble all reactions on ice to avoid premature cDNA synthesis.
- The recommended RNA input is from 1 pg to 100 ng total RNA.
- Prepare a reaction cocktail to reduce pipetting errors. Dispense equal aliquots into reaction tubes. Add RNA to each reaction as a final step. Addition of 2 - 5 μl volumes of RNA will improve assay precision.
- Include a No Template Control (NTC) and No RT Control (NRT) when necessary. The NTC will enable detection of contamination in the reaction components. The NRT control tests for contaminating genomic DNA in the reaction.
- Calculate the required volumes of each component based on the following table:
| Final concentration | 20 μl rxn | |
| Nuclease-free water up to 20 μl | As required | |
| KAPA SYBR® FAST 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 |
| dUTP (10 mM) (optional) | 200 μM | 0.4 μl |
| ROX Reference Dye | Included in master mix at 500 nM final | N/A |
| KAPA RT Mix (50X) | 1X | 0.4 μl |
| Template RNA | variable | < 5 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:
| Step | Temperature | Duration | Cycle |
| cDNA synthesis | 42°C | 5 min | Hold |
| Inactivate RT | 95°C | 2-5 min | Hold |
| Denature | 95°C | 3 sec | 40 |
| Anneal/extend | 60°C | ≥ 20 sec | 40 |
| Dissociation | Accordind to | instrument | guidelines |