Two-Step RT-HDA (Reverse Transcription tHDA)
- Prepare a fresh 7-fold dilution of ThermoScript RT (15 U/μl, Invitrogen, Cat# 12236-022).
- 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 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
- 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.
- 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.
- Incubate at 65ºC for 120 minutes using a thermocycler, a water bath or an incubator.
- 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