Introduction

This Quick Reference Page is provided as a general reference when using the SYBR® GreenER™ Two-Step qRT-PCR Kit for ABI PRISM®. For more detailed protocols and information, refer to the individual product documentation provided in each module box.

Kit Modules

The SYBR® GreenER™ Two-Step qRT-PCR Kit for ABI PRISM® is provided as two separate modules:

  1. Use the SuperScript™ III First-Strand Synthesis SuperMix for qRT-PCR module for first-strand cDNA synthesis.

  2. Use the SYBR® GreenER™ qPCR SuperMix for ABI PRISM® module for qPCR.

Storage

Modules are shipped on dry ice, and should be stored separately as follows:

Module:                                                                                                                                                          Storage:

SuperScript™ III First-Strand Synthesis SuperMix for qRT-PCR                                                                     -20°C
SYBR® GreenER™ qPCR SuperMix for ABI PRISM®                                                                                       4°C

Note

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.
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First-Strand cDNA Synthesis Protocol

Use the reagents provided in the SuperScript™ III First-Strand Synthesis SuperMix for qRT-PCR module to generate first-strand cDNA from up to 1 μg of purified total RNA, as follows:

  1. 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 to20 μl


  2. Gently mix tube contents and incubate at 25°C for 10 minutes.

  3. Incubate tube at 50°C for 30 minutes.

  4. Terminate the reaction at 85°C at 5 minutes, and then chill on ice.

  5. Add 1 μl (2 U) of E. coli RNase H and incubate at 37°C for 20 minutes.

  6. 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).
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qPCR Protocol

Following first-strand cDNA synthesis, use the reagents provided in the SYBR® GreenER™ qPCR SuperMix for ABI PRISM® module to perform qPCR, as follows:

  1. 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

  2. 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 μl1X
    Forward primer, 10 μM1 μl200 nM
    Reverse primer, 10 μM1 μl200 nM
    cDNA template (from Step 6 above)≤5 μl(max. 10% v/v)
    Autoclaved distilled waterto 50 μl

  3. 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.

  4. Place reactions in a preheated thermal cycler programmed as described above. Collect and analyze the results.
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11763.QRC        Version B     13-Dec-2006