PCR Cycle TimerFree PCR timer for timing thermal cycling stages manually. Denaturation: 30s at 95°C. Annealing: 30s. Extension: 1 min/kb. Track your manual water bath PCR protocol.Frequently asked questionsWhat are the three stages of a PCR cycle?1. Denaturation (95°C, 30 seconds): double-stranded DNA separates into single strands. 2. Annealing (50–65°C, 30 seconds): primers bind to complementary sequences on the single-stranded template. 3. Extension (72°C, 1 minute per kb): Taq polymerase synthesises new DNA from the primer. Each cycle doubles the target sequence.How long does PCR take?Standard PCR (30 cycles): 2–3 hours in an automated thermocycler. Initial denaturation (2–5 min) + 30 cycles × (denaturation + annealing + extension time) + final extension (5–10 min). For a 1 kb product with 30-second stages: approximately 2 hours total.How do I calculate extension time for PCR?Use 1 minute per kilobase of target DNA for standard Taq polymerase. For a 2 kb product: 2-minute extension. High-fidelity polymerases (Phusion, Q5): often use 15–30 seconds per kb. Fast PCR protocols use 5–15 seconds per kb with optimised master mixes.What annealing temperature should I use?Start 5°C below the lowest primer Tm (melting temperature). If primer Tms are 60°C and 65°C, try 55°C initially. Too low: non-specific bands. Too high: no product. Gradient PCR (testing multiple temperatures simultaneously) is the fastest way to optimise annealing temperature.How many PCR cycles should I use?25–35 cycles is standard. Under 25: may be insufficient for low-template starting material. Over 35: accumulation of PCR errors from non-specific amplification and Taq error incorporation. For diagnostic PCR from single-copy genomic DNA: 30–35 cycles. For cloning from cDNA: 25–30 cycles.