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  • One-Step RT-HDA (Reverse Transcription tHDA)

    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 0.2 ml or a 0.5 ml micro centrifuge tube 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
      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. Incubate at 65ºC for 120 minutes using a thermocycler, a water bath or an incubator.
    5. Load 10 μl of the RT-HDA product on a 2% agarose gel. 

      * 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