cDNA Synthesis Calculator

🧬 RT‑cDNA calculators NEB & Thermo

ProtoScript® II First Strand cDNA Synthesis  ·  20 µL reactions

Number of reactions exact volumes, no overage

Primer Oligo d(T)₂₃ VN Random Primer Mix

Component	Per rxn	Total

⏱️  Incubate at 42 °C for 1 hour. Oligo d(T)₂₃ VN primer. Inactivate at 80 °C for 5 min.

Based on NEB protocol E6560 • 20 µL reaction • up to 1 µg RNA

SuperScript™ IV First‑Strand Synthesis System  ·  20 µL reactions

Number of reactions exact volumes, no overage

Primer Oligo d(T)₂₀ (50 µM) Random Hexamers (50 ng/µL) Gene‑specific (2 µM)

Template RNA (µL) 10 pg–5 µg total RNA · up to 11 µL

Component	Per rxn	Total

Step 1: Heat RNA‑primer mix at 65 °C for 5 min → ice ≥1 min.
Step 3: Combine annealed RNA with RT reaction mix.
Step 4: Oligo d(T)₂₀ / gene‑specific: 50–55 °C for 10 min. Inactivate at 80 °C for 10 min.
Optional: add 1 µL E. coli RNase H, 37 °C for 20 min to remove RNA.

Based on Thermo Fisher SuperScript™ IV user guide • 20 µL reaction • 10 pg–5 µg total RNA

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