Polymerase Chain Reaction
PCR amplifies specific DNA regions using template, primers, nucleotides, and heat-stable polymerase. Thermal cycling: denaturation (~95°C), annealing (~55–65°C), extension (~72°C).
Primer design
Primers flank target sequence (typically 18–25 bp each). Specificity prevents amplifying wrong regions. BLAST primers against genome to check off-target binding.
Gel electrophoresis
DNA migrates through agarose matrix toward positive electrode — smaller fragments move faster. Stain with safe dyes (e.g., SYBR Safe) and visualize under UV or blue light.
Common issues
- No band — degraded DNA, wrong annealing temperature, or missing reagent.
- Multiple bands — nonspecific priming or contamination.
- Smear — too much template or degraded RNA co-purified.