PCR Calculator
Plan your PCR setup with this calculator. Estimate optimal annealing temperature and reagent volumes for accurate polymerase chain reaction protocols.
Annealing Temperature Estimation
Optimal Annealing Temperature:
55°CFormula: (Tm Forward + Tm Reverse) / 2 - 5°C
Reagent Concentration
PCR Reaction Mix Calculator
Reagent Volumes:
| Component | Volume |
|---|---|
| 10X PCR Buffer | 5.0 μL |
| dNTPs (0.8 mM total) | 1.0 μL |
| MgCl₂ (25 mM) | 3.0 μL |
| Forward Primer (10 μM) | 5.0 μL |
| Reverse Primer (10 μM) | 5.0 μL |
| DNA Template | 1-5 μL |
| DNA Polymerase (5 U/μL) | 0.2-0.5 μL |
| Nuclease-free Water | to 50 μL |
PCR Cycling Conditions
- Initial Denaturation: 95°C for 2-5 minutes
- 25-35 Cycles of:
- Denaturation: 95°C for 30 seconds
- Annealing: 55°C for 30 seconds
- Extension: 72°C for 1 minute per kb
- Final Extension: 72°C for 5-10 minutes
- Hold: 4°C
Common Mistakes in PCR Setup
Incorrect Annealing Temperature
Setting annealing temperature too high prevents primer binding, while too low allows non-specific binding. Aim for 5-10°C below the lowest primer Tm.
Imbalanced Mg²⁺ Concentration
Mg²⁺ is a critical cofactor for DNA polymerase. Too little reduces yield, while excess promotes non-specific amplification. Optimal range is typically 1.5-3 mM.
Template Quality Issues
Poor quality or quantity of template DNA can lead to failed amplification. Ensure DNA is pure and use 1-10 ng for plasmid DNA or 10-100 ng for genomic DNA.
Incompatible Primer Pair
Primers with significantly different Tm values create amplification bias. Keep Tm difference within 5°C and avoid secondary structures or primer dimers.