PCR Optimization Calculator

What is PCR Optimization?

PCR optimization involves adjusting reaction conditions to achieve specific and efficient amplification. Key parameters include annealing temperature, primer design, and cycling conditions.

How to Use This Calculator

1. Enter your primer sequences
2. Specify salt and primer concentrations
3. Input template characteristics
4. Get optimized conditions instantly

When to Use

• Setting up new PCR reactions
• Troubleshooting failed amplifications
• Optimizing multiplex PCR
• Calculating master mix volumes

Tm Calculation Methods

For primers < 14 nt: Basic Tm = 4(G+C) + 2(A+T)

For primers ≥ 14 nt: Salt-adjusted formula with nearest-neighbor parameters

Optimization Tips

• Annealing temp: Start at Tm - 5°C
• Primer Tm difference: Keep < 5°C
• Extension: 1 min/kb for Taq
• Cycles: 25-35 for most applications

FAQ

Q: What if primer Tms differ significantly?
A: Use gradient PCR or redesign primers for similar Tms.

Q: How many cycles should I use?
A: 25-30 for abundant templates, 30-35 for rare templates.