PCR Amplification Calculator

Calculate theoretical DNA yield and amplification efficiency for your PCR reactions

PCR Parameters

100% = perfect doubling each cycle (theoretical maximum)
Used for molecular weight calculations

Amplification Results

Enter your PCR parameters and click "Calculate Amplification" to analyze your reaction.

Perfect for planning experiments and predicting yields.

Let's Understand PCR Amplification

PCR (Polymerase Chain Reaction) is a revolutionary molecular biology technique that amplifies specific DNA sequences exponentially. Understanding the mathematics behind PCR amplification is crucial for optimizing reactions and predicting yields in research and diagnostics.

DNA Amplification
Exponential increase in target DNA
Yield Prediction
Calculate theoretical outcomes
Optimization
Improve reaction efficiency

PCR Mathematics & Theory

Exponential Phase Formula

Nf = N0 × (1 + E)n
  • Nf = Final DNA amount
  • N0 = Initial template amount
  • E = PCR efficiency (0-1)
  • n = Number of cycles

This formula assumes optimal conditions where DNA doubles each cycle. Real-world efficiency typically ranges from 80-100%.

PCR Efficiency Calculation

E = (10-1/slope - 1) × 100%
Efficiency Ranges:
  • 90-100% - Excellent
  • 80-90% - Good
  • 70-80% - Acceptable
  • <70% - Poor (needs optimization)

Copy Number Conversion

Copies = (Mass × 6.022×10²³) / (Length × 650 × 2)

Key Constants:

  • 650 Da = Average molecular weight per bp
  • 1 ng dsDNA ≈ 3 × 10¹¹ bp
  • Human genome ≈ 3.2 × 10⁹ bp
  • E. coli genome ≈ 4.6 × 10⁶ bp

Plateau Phase Modeling

After ~30-35 cycles, amplification efficiency decreases due to:

  • Reagent depletion (dNTPs, primers)
  • Product inhibition
  • Polymerase degradation
  • Template secondary structures
Tip: For quantitative PCR, stay in exponential phase (Cq < 35)

PCR Workflow & Temperature Cycling

Step 1

Denaturation

94-98°C
30 seconds - 2 minutes

High temperature separates double-stranded DNA into single strands by breaking hydrogen bonds between base pairs.

Step 2

Annealing

50-65°C
15-60 seconds

Primers bind to their complementary sequences on the single-stranded DNA template. Temperature depends on primer Tm.

Step 3

Extension

72°C
1 min per kb

DNA polymerase synthesizes new DNA strands from the primers. Taq polymerase optimal temperature. 1 kb/min synthesis rate.

Factors Affecting PCR Efficiency

Template Quality

  • DNA Purity: A260/A280 ratio 1.8-2.0
  • Integrity: Avoid degraded templates
  • Concentration: 1 ng - 1 μg per reaction
  • Secondary Structure: GC-rich regions
  • Inhibitors: Phenol, EDTA, salt excess

Primer Design

  • Length: 18-25 nucleotides
  • Tm: 55-65°C, within 2°C of each other
  • GC Content: 40-60%
  • Specificity: Avoid secondary structures
  • Concentration: 0.1-1.0 μM final

Reaction Components

  • dNTPs: 200 μM each (A, T, G, C)
  • MgCl₂: 1.5-3.0 mM (enzyme cofactor)
  • Buffer: pH 8.3-8.8, ionic strength
  • Polymerase: 0.5-2.5 U per reaction
  • Additives: BSA, DMSO, betaine

Cycling Conditions

  • Cycle Number: 25-40 cycles optimal
  • Ramp Rates: 2-5°C/second
  • Hold Times: Optimize for amplicon
  • Hot Start: Prevents non-specific priming
  • Final Extension: 5-10 min at 72°C

PCR Applications & Variants

Quantitative PCR (qPCR)

Real-time monitoring of PCR amplification using fluorescent reporters.

  • Gene expression analysis
  • Viral load quantification
  • Copy number variation
  • Food pathogen detection
  • Environmental monitoring

Reverse Transcription PCR

Amplification of RNA targets via cDNA synthesis step.

  • mRNA expression studies
  • Viral RNA detection
  • microRNA analysis
  • COVID-19 testing
  • Cancer biomarkers

Digital PCR

Absolute quantification through sample partitioning.

  • Rare allele detection
  • CNV analysis
  • NGS library quantification
  • GMO detection
  • Minimal residual disease

Multiplex PCR

Simultaneous amplification of multiple targets.

  • STR profiling
  • Pathogen panels
  • Genetic disorder screening
  • Pharmacogenomics
  • Forensic identification

PCR Troubleshooting Guide

No Amplification

Possible Causes:
  • Degraded template DNA
  • Inactive polymerase
  • Primer degradation
  • Incorrect cycling conditions
  • Inhibitors in sample

Non-Specific Products

Solutions:
  • Increase annealing temperature
  • Use hot-start polymerase
  • Optimize primer concentration
  • Redesign primers
  • Add specificity enhancers

Low Efficiency

Optimization Steps:
  • Check Mg²⁺ concentration
  • Optimize primer ratios
  • Adjust cycling parameters
  • Use PCR enhancers
  • Fresh reagents

Optimization Tips

Best Practices:
  • Gradient PCR for optimization
  • Positive/negative controls
  • Store reagents properly
  • Use certified tubes
  • Regular calibration

Practical Calculation Examples

Example 1: Standard PCR

Initial Template: 10 ng genomic DNA
Cycles: 30
Efficiency: 95%
Target: 500 bp fragment
Amplification = (1 + 0.95)³⁰
= 1.95³⁰ ≈ 637,621,500
Final Yield ≈ 6.4 μg

Example 2: qPCR Analysis

Initial Copies: 1000 copies
Cq Value: 25 cycles
Efficiency: 90%
Detection Threshold: 10⁶ copies
Amplification = (1 + 0.90)²⁵
= 1.90²⁵ ≈ 72,765,000
Final Copies ≈ 7.3 × 10¹⁰

Quick Reference Guide

Typical Concentrations
  • Template: 1 ng - 1 μg
  • Primers: 0.1 - 1.0 μM
  • dNTPs: 200 μM each
  • MgCl₂: 1.5 - 3.0 mM
  • Taq: 1.25 - 2.5 U
Cycling Parameters
  • Initial: 95°C, 5 min
  • Denature: 95°C, 30 s
  • Anneal: Tm-5°C, 30 s
  • Extend: 72°C, 1 min/kb
  • Final: 72°C, 10 min
Quality Metrics
  • Efficiency: 90-110%
  • R²: > 0.995
  • Slope: -3.1 to -3.6
  • Y-intercept: < 40
  • Cq CV: < 5%

Related Biology Calculators

Loading related calculators.

Ratings

Ready to Explore More Biology Calculators?

Discover our complete range of biology calculators to help with your research and education.

View All Biology CalculatorsExplore Other Categories