Two-Step RT-HDA (Reverse Transcription tHDA)

Protocol

1. Prepare a fresh 7-fold dilution of ProtoScript® II RT (NEB #M0368).
2. To set up a 50 μl RT-HDA reaction, prepare a 25 μl Mix A in a 0.5 ml microcentrifuge tube in a sterile hood or a PCR Workstation:

   H2O	X μl
   10X Annealing buffer II	2.5 μl
   RNA 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 μl 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
   ProtoScript® II RT (2.0 U/μl)*	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 conditions of tHDA reactions can be further optimized by titering the following 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