The SYBR® GreenER™ Two-Step qRT-PCR Kit for ABI PRISM® is provided as two separate modules:
- Use the SuperScript™ III First-Strand Synthesis SuperMix for qRT-PCR module for first-strand cDNA synthesis.
- Use the SYBR® GreenER™ qPCR SuperMix for ABI PRISM® module for qPCR.
Modules are shipped on dry ice, and should be stored separately as follows:
SuperScript™ III First-Strand Synthesis SuperMix for qRT-PCR -20°C
SYBR® GreenER™ qPCR SuperMix for ABI PRISM® 4°C
This kit is designed for use with real-time ABI instruments that are compatible with ROX Reference Dye at a final concentration of 500 nM. These instruments include the ABI PRISM® 7000, 7700, and 7900HT; the ABI 7300 Real-Time PCR System; and the ABI GeneAmp® 5700. This kit is not compatible with instruments that use ROX at a final concentration lower than 500 nM, including the ABI 7500.
- Combine the following kit components in a tube on ice. For multiple reactions, a master mix without RNA may be prepared:
2X RT Reaction Mix (includes oligo(dT)20 (2.5 μM), random hexamers (2.5 ng/μl), 10 mM MgCl2, and dNTPs) 10 μl RT Enzyme Mix (includes SuperScript™ III RT and RNaseOUT™) 2 μl RNA (up to 1 μg total RNA) x μl DEPC-treated water to 20 μl
- Gently mix tube contents and incubate at 25°C for 10 minutes.
- Incubate tube at 50°C for 30 minutes.
- Terminate the reaction at 85°C at 5 minutes, and then chill on ice.
- Add 1 μl (2 U) of E. coli RNase H and incubate at 37°C for 20 minutes.
- Use undiluted or diluted cDNA in the qPCR protocol below, or store the reaction at -20°C until use.
Note: Up to 10% of the qPCR reaction volume may be undiluted cDNA (e.g., for a 50-μl qPCR, use up to 5 μl of undiluted cDNA).
- Program the ABI real-time instrument to perform a brief UDG incubation immediately followed by PCR amplification, as shown below. Optimal cycling temperatures and times may vary for different target sequences, primer sets, and instruments.
50°C for 2 minutes hold (UDG incubation)
95°C for 10 minutes hold
40 cycles of:
95°C, 15 seconds
60°C, 60 seconds
Melting curve analysis: Refer to instrument documentation
- For each reaction, add the following to a 0.2-ml microcentrifuge tube or each well of a PCR plate. A standard 50-μl reaction size is provided below; component volumes can be scaled as desired (e.g., scaled down to a 20-μl reaction volume for 384-well plates).
For multiple reactions, prepare a master mix of common components, add the appropriate volume to each tube or plate well, and then add the unique reaction components (e.g., template). Note: Preparing a master mix is strongly recommended in qRT-PCR to reduce pipetting errors.
Component Amount Final Conc. SYBR® GreenER™ qPCR SuperMix for ABI PRISM® 25 μl 1X Forward primer, 10 μM 1 μl 200 nM Reverse primer, 10 μM 1 μl 200 nM cDNA template (from Step 6 above) ≤5 μl (max. 10% v/v) Autoclaved distilled water to 50 μl
- Cap or seal the reaction tube/PCR plate, and gently mix. Make sure that all components are at the bottom of the tube/plate; centrifuge briefly if needed.
- Place reactions in a preheated thermal cycler programmed as described above. Collect and analyze the results.