First Strand cDNA Synthesis (AB Kit)


Materials

Protocol

  • Generate Master Mix Containing:
Master Mix Components
Component Per Reaction Per 10 Reactions
10X RT Buffer 2 20
25X dNTP Mix (100 mM) 0.8 8
10X RT Random Primers 2 20
MultiScribe Reverse Transcriptase 1 10
Nuclease Free Water 4.2 42
  • Add 10 uL Master mix per well into tube or plate
  • Add 10 ug RNA in a final volume of 20 uL to each well. Since results will be normalized in the end, standardizing RNA quantities at this step is unnecessary. Total volume can be adjusted down if RNA is in short supply.
  • Run the PCR Program AB Reaction Protocol. Takes ~2h:
    • 10 min at 25C
    • 120 min at 37C
    • 5 min at 85C