One-Step RT-HDA (Reverse Transcription tHDA)
Protocol
- 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.
- 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.
- Mix the reaction by brief vortexing or by pipetting followed by brief centrifugation.
- Overlay the reaction mixture with 50 μl min eral oil.
- Incubate 42°C for 2 minutes; then immediately transfer to 65ºC water 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 ProtoScript II RT (NEB M0368S) 1 to 2 units 0.5 to 10.5 units
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 |