PCR Amplification — Standard Protocol
Quick run (TL;DR)
- Prepare master mix as listed below.
- Thermocycler: 98 °C 30 s; 30 cycles (98 °C 10 s, 60 °C 20 s, 72 °C 30 s/kb); 72 °C 2 min.
- Run 1–2 % agarose gel; visualize product.
- Document results; proceed to cleanup if single band.
- Store product at −20 °C.
Materials
- High‑fidelity polymerase master mix
- Primers (10 µM each)
- Template DNA
- Nuclease‑free water
Steps
Step 1 — Master mix preparation
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Step 2 — Thermocycling
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Step 3 — Gel electrophoresis
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Troubleshooting
- No band: increase annealing time or adjust Tm ±2 °C.
- Multiple bands: raise annealing temperature; redesign primers.
References
- Lorem ipsum reference 1.
- Lorem ipsum reference 2.
Cross-links
Referenced in notebook: Week 2025‑08‑15 — PCR test
