One-Step RT-HDA (Reverse Transcription tHDA)


  1. Prepare a fresh 100-fold dilution of ProtoScript II Reverse Transcriptase (200 U/μl, NEB M0368S) in 1X ProtoScript II buffer. Keep on ice. Discard unused dilution.
  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.
  3. Nuclease-free H2O (NEB #1500S) 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
    RNA template X μl
    Forward Primer (5 μM)* 0.75 μl
    Reverse Primer (5 μM)* 0.75 μl
    IsoAmp® Enzyme Mix 3.5 μl
    ProtoScript II RT (2.0 U/μl) 1.0μl
    Total volume 50 μl
  4. Mix the reaction by brief vortexing or by pipetting followed by brief centrifugation.
  5. Overlay the reaction mixture with 50 μl min eral oil.
  6. Incubate 42°C for 2 minutes; then immediately transfer to 65ºC water for 120 minutes using a thermocycler, a water bath or an incubator.
  7. 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
    ProtoScript II RT (NEB M0368S) 1 to 2 units 0.5 to 10.5 units