• My NEB
  • Print
  • PDF
  • Two-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. To set up a 50 ìl RT-HDA reaction, prepare a 25 ìl Mix A in a 0.5 ml micro centrifuge tube in a sterile hood or a PCR Workstation:
      H2O X μl
      10X Annealing buffer II 2.5 μl
      DNA template X μl
      Forward Primer (5 μM)* 0.75 μl
      Reverse Primer (5 μM)* 0.75 μl
      Total volume of Mix A 25 μl
      In addition, prepare a 25 ìl Mix B in a separate 0.5-ml microcentrifuge tube in a sterile hood or a PCR Workstation:
      H2O 9.25 μl
      10X Annealing buffer II 2.5 μl
      MgSO4 (100 mM)* 1.75 μl
      NaCl (500 mM)* 4 μl
      IsoAmp® dNTP Solution 3.5 μl
      IsoAmp® Enzyme Mix 3.5 μl
      ThermoScript RT (2.1 U/μl, Invitrogen)* 0.5 μl
      Total volume of Mix B 25 μl
    3. Gently mix each of the mixes by brief vortexing or by pipetting followed by brief centrifugation. Overlay the reaction mixture with 50 μl mineral oil. Place the tubes on ice.
    4. Incubate Mix A at 95ºC for 2 minutes and place promptly on ice. Add 25 μl of Mix B into Mix A underneath the oil layer and gently mix the reaction by pipetting. Place the tubes on ice.
    5. Incubate at 65ºC for 120 minutes using a thermocycler, a water bath or an incubator.
    6. 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