PCR Primer Stats

Analyze PCR primer properties including melting temperature, GC content, and PCR suitability.

Tool Configuration
Configure the parameters for PCR Primer Stats

Paste one or more FASTA sequences (max 5,000,000 characters, max primer length 50 bases).

1

Input Primer Sequences

Paste one or more primer sequences in FASTA format. Primers should be 1-50 bases long. The tool accepts multiple primers for batch analysis, making it ideal for evaluating primer pairs or primer libraries.

2

Understanding Tm Calculations

Three melting temperature methods are provided: Basic Tm (simple formula), Salt-adjusted Tm (considers ionic strength), and Nearest-neighbor Tm (most accurate, considers sequence context). Use nearest-neighbor Tm for precise PCR optimization.

3

Evaluating Suitability Tests

The tool checks for common primer design problems: base runs (repetitive sequences), length (14-30 bp recommended), GC content (40-60% optimal), Tm range (50-58°C suitable), GC clamp (1-3 G/C at 3' end), self-annealing, and hairpin formation. Warnings indicate potential PCR problems.

4

Practical Applications

Use this tool to validate primer designs before ordering, troubleshoot failed PCR reactions, compare multiple primer candidates, and ensure primer pairs have matched Tm values. The molecular weight and concentration data help with primer dilution calculations.

PCR Primer Analysis
Information about primer property calculations and suitability criteria.

Melting Temperature (Tm)

Three Tm calculation methods are provided:

  • Basic Tm: Simple calculation based on base composition
  • Salt-adjusted Tm: Takes into account salt concentration
  • Nearest-neighbor Tm: Most accurate, considers sequence and base stacking

PCR Suitability Criteria

Primers are evaluated for:

  • Single base runs (max 4 consecutive identical bases)
  • Dinucleotide runs (max 4 consecutive repeats)
  • Length (14-30 bases recommended)
  • GC content (40-60% optimal)
  • Tm range (50-58°C suitable)
  • GC clamp (1-3 G/C at 3' end)
  • Self-annealing and hairpin formation