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  • One-Step qRT-HDA (Real-time quantitative RT-HDA)

    Overview

    The following protocol is intended for real-time detection using the Applied Biosystems 7300 Real-Time PCR System. 

    The kit also can be coupled with real-time detection methods to conduct realtime quantitative tHDA (qHDA) and RT-HDA (qRT-HDA) to monitor amplification as it progresses. For optimal performance, use EvaGreen as a reporter dye and ROX as a passive reference dye. Sequence-specific probes can also be designed for qHDA experiments.

    Protocol

    1. Prepare a fresh 7-fold dilution of ThermoScript RT (15 U/μl, Invitrogen, Cat# 12236-022).
    2. Set up a 50 μl reaction in a MicroAmp optical tube (Applied BioSystems) in a sterile hood or a PCR Workstation.
      H2O X μl
      10X Annealing buffer II 5 μl
      MgSO4 (100 mM)* 1.75 μl
      NaCl (500 mM)* 4 μl
      IsoAmp® dNTP Solution 3.5 μl
      DNA template X μl
      Forward Primer (5 μM)* 0.75 μl
      Reverse Primer (5 μM)* 0.75 μl
      IsoAmp® Enzyme Mix 3.5 μl
      ThermoScript RT (2.1 U/ìl, Invitrogen)* 0.5 μl
      EvaGreen (20X, Biotium) 0.5 μl
      ROX Reference Dye (50X, Invitrogen) 1 μl
      Total volume 50 μl
    3. Mix the reaction by brief vortexing or by pipetting followed by brief centrifugation. Overlay the reaction mixture with 50 μl min eral oil. Place the tubes on ice.
    4. Real-time detection is carried out on a 7300 Real-Time PCR System (ABI) with the following well inspector setting: reporter dye: SYBR; quencher: none; passive reference dye: ROX. Use the following program:
      Stage 1: (60 X)
        Step1: 66ºC for 0:05
        Step2: 65ºC for 1:55
        Data collection and real-time analysis enabled
      Stage 2: (1 X)
        Dissociation Stage (default settings of the machine)
        Melt curve data collection and analysis enabled

      * The condition of tHDA reactions can be further optimized by titering thefollowing components:
      Components Recommended concentration Recommended concentration for titering
      MgSO4 3.5 to 4 mM 3 to 4.5 mM
      NaCl 30 to 40 mM 20 to 50 mM
      Primer 75 to 100 nM 50 to 200 nM
      ThermoScript RT 1 to 2 units 0.5 to 10.5 units