Before you start

Validate primers: one melt peak, efficiency 90–110% from a standard curve, intron-spanning where possible.
Plan the plate: triplicates, no-template controls and no-RT controls.

Reagents and materials

Totals are for 96 wells (+10% extra). Per-well (10 µL) amounts are in the middle column.

Reagent	Per well (10 µL)	Total
2× SYBR Green master mix	5 µL	5 µL
Forward primer, 10 µM (0.3 µM final)	0.3 µL	0.3 µL
Reverse primer, 10 µM (0.3 µM final)	0.3 µL	0.3 µL
Nuclease-free water	2.4 µL	2.4 µL
Diluted cDNAadded separately	2 µL	2 µL

ΔCt (each sample)	Ct(target) − Ct(reference gene)
ΔΔCt	ΔCt(sample) − mean ΔCt(control group)
Fold change	2^(−ΔΔCt)
Statistics	Test on ΔCt (or −ΔΔCt) values, not on fold change

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

Make primer master mixes
For each gene, combine SYBR mix, primers and water for all wells plus 10% extra.
Load the plate
Dispense 8 µL master mix per well, then 2 µL cDNA. Seal firmly.
Spin
Centrifuge the plate 1 min at ~1,000 × g to remove bubbles.
Run
Typical cycling: 95 °C 2–10 min (enzyme activation); 40 cycles of 95 °C 15 s and 60 °C 60 s; then a melt curve.
Check quality
Confirm single melt peaks, flat no-template controls and replicate Ct spread under ~0.3 cycles.
Calculate
Average technical replicates, calculate ΔCt against the reference gene(s), then ΔΔCt and fold change.

Use one or more stable reference genes, validated for your conditions.
ΔΔCt assumes similar efficiency for target and reference; otherwise use an efficiency-corrected method (Pfaffl).
Biological replicates (animals, cultures) — not wells — are your n.

Problem	What to check or change
Multiple melt peaks	Primer-dimers or non-specific product: redesign primers or raise annealing temperature.
Amplification in NTC	Fresh water and primers; separate pre- and post-PCR areas.
Ct > 35	Low expression or input: use more cDNA, check RT.

Standard laboratory care.