Before you start

Make a master mix for all reactions plus 10% extra.
Controls: known wild-type, heterozygous and homozygous DNA, and a no-template control.

Reagents and materials

Totals are for 24 reactions (+10% extra). Per-reaction (20 µL) amounts are in the middle column.

Reagent	Per reaction (20 µL)	Total
2× PCR master mix (Taq, dNTPs, MgCl₂, buffer)	10 µL	10 µL
Forward primer, 10 µM (0.5 µM final)	1 µL	1 µL
Reverse primer, 10 µM (0.5 µM final)	1 µL	1 µL
Nuclease-free water	6 µL	6 µL
Genomic DNA templateadded separately to each tube	2 µL	2 µL

Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.

Make the master mix
Combine master mix, primers and water on ice; vortex briefly and spin down.
Dispense
Add 18 µL master mix to each tube, then 2 µL template; mix and spin.
Cycle
Typical program: 94–95 °C 3 min; 35 cycles of 94 °C 30 s, 55–62 °C 30 s (primer-specific), 72 °C 30–60 s per kb; final 72 °C 5 min; hold at 4 °C.
Run a gel
Load 10 µL on a 1.5–2% agarose gel with a ladder and run until bands separate.
Call genotypes
Compare band sizes with your controls and the expected sizes for each allele.

Use the annealing temperature validated for your primer set (often from the strain’s published protocol).
Set up reactions in a separate area from gel/PCR product handling.

Problem	What to check or change
No bands	Check template amount, primer integrity, MgCl₂, annealing temperature (try a gradient).
Extra bands	Raise the annealing temperature, lower cycle number or template.
Band in no-template control	Replace water and primers; clean pipettes; use filter tips.

Standard laboratory care.