💡 Quick Summary

PCR Primer Stats analyses a forward and reverse primer pair. It returns melting temperature (three methods), %GC, molecular weight, extinction coefficients, and eight PCR suitability checks for each primer, plus three pair compatibility tests: Tm difference, heterodimer potential, and 3′-end complementarity.

📋 How to Use

Paste the forward primer sequence (5′→3′) into the Forward Primer field. Raw sequence or FASTA format accepted.
Paste the reverse primer sequence (5′→3′) into the Reverse Primer field. Enter it exactly as you would order it — do not reverse-complement it.
Click Submit to run the analysis. You will get individual reports for each primer and a pair compatibility card.
Click Load Example to analyse the pGEX forward/reverse cloning primer pair.
Use Copy All to copy the full text report to your clipboard.

🧮 Formulas & Logic

Basic Tm

If length < 14 bp: 4×(G+C) + 2×(A+T). Otherwise: 64.9 + 41×(G+C−16.4) / length (Rychlik & Rhoads 1989)

Salt adjusted Tm

81.5 + 7.21×ln([salt]) + 0.41×%GC − 675/length (Sambrook et al. 1989)

Nearest neighbor Tm

Tm = (1000×ΔH) / (ΔS + R×ln(C_T/2)) − 273.15 (SantaLucia 1998; von Ahsen et al. 1999)

Molecular weight

G×329.21 + A×313.21 + T×304.2 + C×289.18 − 61.96 (Da)

Extinction coefficient

2×Σ(dimer) − Σ(internal single) (NetPrimer coefficients)

Heterodimer

Forward primer aligned against reverse(Reverse primer) using complementarity scoring (G↔C, A↔T). Flags contiguous complementary bases.

3′-end compatibility

Last 5 bases of each primer aligned for complementarity. Flags potential 3′ primer-dimer formation.

Tm difference

ΔTm = |Tm(forward) − Tm(reverse)| using nearest-neighbor Tm. Pass if ≤ 5 °C.

📊 Result Interpretation

Basic Tm

Fast estimate; accurate for short primers (< 14 bp)

Salt adjusted Tm

Accounts for monovalent cation concentration; good general estimate

Nearest neighbor Tm

Most accurate; accounts for base stacking, salt (50 mM K⁺/Na⁺ + 1.5 mM Mg²⁺), and primer concentration (200 nM)

Single base runs

Pass if no 5+ consecutive identical bases (e.g. GGGGG)

Dinucleotide runs

Pass if no 5+ consecutive identical dinucleotides (e.g. GAGAGA)

Length

Pass if 14–30 bases

Percent GC

Pass if 40–60%

Tm (Nearest neighbor)

Pass if 50–58 °C

GC clamp

Pass if 1–3 G/C in last 5 bases

Self-annealing

Pass if ≤ 3 contiguous self-complementary bases and ≤ 50% self-annealing

Hairpin formation

Pass if ≤ 3 contiguous hairpin bases and ≤ 50% in a hairpin

Tm difference

Pass if ΔTm ≤ 5 °C. Large differences reduce amplification efficiency when both primers are in the same reaction.

Heterodimer potential

Pass if ≤ 3 contiguous cross-complementary bases and ≤ 50% cross-annealing.

3′-end compatibility

Pass if ≤ 3 complementary bases in the last 5 positions of each primer. 3′ primer-dimers can be extended by polymerase and reduce yield.

🔬 Applications

Designing primer pairs with matched melting temperatures for optimal PCR
Detecting potential primer-dimer formation between forward and reverse primers
Checking 3′-end complementarity that could cause extendable primer-dimers
Evaluating self-annealing and hairpin structure for individual primers
Verifying GC content, GC clamp, and appropriate primer length before ordering

⚠️ Common Mistakes & Warnings

Primers > 50 bases are rejected

Each primer must be ≤ 50 bp. Longer sequences will produce an error.

Reaction conditions are fixed

Nearest neighbor Tm uses fixed settings: 50 mM salt, 1.5 mM Mg²⁺, 200 nM primer, no 5′-phosphate. These match the original SMS defaults.

Enter the reverse primer 5′→3′

Paste the reverse primer in the standard synthesis orientation (5′ to 3′), exactly as you would order it. The tool reverse-complements it internally for heterodimer and 3′-end checks.

Degenerate bases show ranges

For primers containing IUPAC degenerate bases (R, Y, S, W, K, M, B, D, H, V, N), all properties are reported as lower-to-upper bounds.

❓ Frequently Asked Questions

Which Tm method should I trust most?

Nearest neighbor Tm is the most accurate because it accounts for base-stacking thermodynamics and actual reaction conditions. Basic Tm is a quick approximation only.

In what orientation should I enter the reverse primer?

Enter the reverse primer in the standard 5′→3′ synthesis orientation — exactly as you would order it from a supplier. The tool handles the necessary reverse-complementation internally for the heterodimer and 3′-end compatibility checks.

What does the heterodimer diagram show?

The forward primer is aligned against the reverse complement of the reverse primer to find complementary regions. Vertical bars (|) mark complement pairs. More than 3 consecutive bars is flagged as a warning.

What is the 3′-end compatibility check?

The tool aligns the last 5 bases of each primer for complementarity. A 3′ primer-dimer has a free 3′-OH that DNA polymerase can extend, consuming primers and reducing PCR yield.

Why do some primers show ranges (e.g. "52 to 58")?

When IUPAC degenerate bases are present, every property is calculated for both the lowest-GC and highest-GC interpretation of the ambiguous bases, giving a min-to-max range.