Primer Design Suite · tools.omics.bd

1 Mode & template

Standard PCR RT / qPCR Inverse PCR Nested PCR Analyse primers

Template sequence (raw or FASTA)

Only A/C/G/T are used; ambiguity codes are converted to N. For inverse PCR, provide the full circular sequence.

2 Target region

Target start (1-based)

Target end (1-based)

Forward primers are placed upstream of the target, reverse primers downstream. Leave blank to scan the whole template.

3 Parameters

Primer length (nt)

Minimum

Optimum

Maximum

Melting temperature (°C)

Minimum

Optimum

Maximum

Tm calculation model

GC content (%)

Minimum

Optimum

Maximum

3′ end & sequence constraints

GC clamp — min G/C in last 5 nt

GC clamp — max G/C in last 5 nt

Max homopolymer run

Max ΔTm between F/R (°C)

Require G or C at 3′ terminus Forbid T at 3′ terminus

Polymerase

Buffer composition

Na⁺ / K⁺ (mM)

Mg²⁺ (mM)

dNTPs (mM total)

Primer conc. (nM)

Free Mg²⁺ is estimated as [Mg²⁺] − [dNTP]; the monovalent equivalent [Na⁺]_eq = [Na⁺] + 4·√[Mg²⁺]_free.

qPCR probe (TaqMan, optional) Design a TaqMan hydrolysis probe for the top-ranked pair

Probe length min

Probe length max

Probe Tm min (°C)

Probe Tm max (°C)

Probe GC min (%)

Probe GC max (%)

Probe max run

Probes to show

A 5′ G on the probe is disallowed (it quenches the reporter). The probe should sit between the primers, with a Tm 8–10 °C above the primers.

Amplicon size (bp)

Minimum

Maximum

Approx. target size (optional)

Tolerance (± bp)

Results to return

Target size is a soft preference — primer pairs closest to it rank first. Typical: RT/qPCR 70–200 bp · Standard PCR 200–1000 bp · Cloning 500–3000 bp.

Forward primer 5′ modification / tail

Preset

Custom 5′ sequence

Reverse primer 5′ modification / tail

Preset

Custom 5′ sequence

The tail is not included in the Tm/GC calculation. It is shown separately in the results (amber).

Stringency

Threshold level

Position step (bp)

Forbidden motifs

Sequences to avoid (comma- or space-separated)

i Notes on the calculations

Tm — SantaLucia (1998) NN parameters with initiation terms; salt correction per SantaLucia or Owczarzy.
ΔG₃₇ — ΔH − TΔS at 298.15 K.
Hairpin — best stem of ≥ 3 consecutive bp with loop ≥ 3 nt.
Self- / cross-dimer — antiparallel ungapped alignments, most stable contiguous run of ≥ 3 bp.
3′-end stability — ΔG of the terminal 5 nt.
No BLAST-level specificity check is performed. Always verify with Primer-BLAST.
For RT/qPCR, place at least one primer or the probe across an exon–exon junction.

📘 Tutorial & Result Interpretation

📑 On this page

Primer design basics — 2-minute overview
The golden rules — what makes a good primer
Choosing the right mode (PCR / qPCR / Inverse / Nested)
Reading Tm, GC and ΔG values
Reading the primer pair table
Secondary structures — hairpin, self-dimer, cross-dimer
TaqMan probe design & interpretation
Nested PCR — outer and inner pairs
Primer analysis mode
5′ tails and modifications
Common pitfalls & troubleshooting

🧬 1. Primer design basics

A primer is a short single-stranded DNA oligo (typically 18–24 nt) that anneals to a template and provides a 3′-OH for a DNA polymerase to extend. Two primers — one on each strand — amplify the region between them.

Forward primer — binds the anti-sense strand and synthesises the sense strand.
Reverse primer — binds the sense strand and synthesises the anti-sense strand.
Amplicon — the DNA product between the two primers, including the primer sequences.

Key rule: the two primers must bind opposite strands and point toward each other so the polymerase copies the region between them.

📏 2. The golden rules

Property	Recommended range	Why
Length	18–24 nt (optimum 20)	Balance between specificity and cost.
GC content	40–60 %	Stable annealing without secondary structure.
Tm (NN)	57–63 °C (optimum 60)	Annealing temperature can be uniform.
ΔTm F vs R	≤ 3 °C	Both primers anneal in the same cycle.
3′ end	1–3 G/C in last 5 nt	Stable 3′ end → efficient extension.
Homopolymer	≤ 4 consecutive same base	Prevents mispriming and slippage.
Hairpin ΔG	> −3 kcal/mol	Prevents self-folding.
Self-dimer ΔG	> −6 kcal/mol	Prevents self-annealing.
Amplicon	100–1000 bp (standard) · 70–200 bp (qPCR)	Efficient amplification.

🎯 3. Choosing the right mode

Mode	When to use	What is different
Standard PCR	Amplify a DNA fragment for cloning, sequencing or genotyping.	Whole-template scan, 200–1000 bp typical amplicon.
RT / qPCR	Quantify expression (mRNA) or detect a target with real-time fluorescence.	Short amplicon (70–200 bp), optional TaqMan probe, exon–exon junction preference.
Inverse PCR	Amplify the sequence flanking a known region — e.g. to find the insertion site of a transposon.	Template is treated as circular; primers point outward into the unknown region.
Nested PCR	Increase specificity when the template is scarce or contaminated.	Two pairs: an outer pair amplifies a large product; an inner pair re-amplifies within it.
Primer analysis	Check primers you already have — Tm, dimers, hairpins.	Input = primer sequences only.

🌡️ 4. Reading Tm, GC and ΔG

Tm (melting temperature)

The temperature at which half the primer is bound to its target. Higher Tm = stronger binding. This tool offers four models:

Model	When it is appropriate
SantaLucia (NN)	Default for primers ≥ 14 nt; uses the nearest-neighbour thermodynamic parameters.
Owczarzy	Same NN base, plus a salt correction for mixed Na⁺/Mg²⁺ buffers. Best when [Mg²⁺] > 1 mM.
Schildkraut	Empirical formula; good for long probes > 40 nt.
Wallace	Simple 2·(A+T) + 4·(G+C); use only for primers < 14 nt.

For routine PCR, stick with SantaLucia — it matches what most commercial primer tools report.

GC content

GC%	Interpretation	Action
40–60 %	Ideal.	Proceed.
30–40 or 60–70 %	Acceptable with caution.	Prefer another candidate if one exists.
< 30 or > 70 %	Weak binding (AT-rich) or secondary structure risk (GC-rich).	Avoid if possible.

ΔG₃₇ (Gibbs free energy)

A more negative ΔG means a more stable structure. This tool reports:

Whole primer ΔG — the overall duplex stability.
3′-end ΔG — stability of the last five bases. Too negative → mispriming; too positive → poor extension.
Hairpin / self-dimer ΔG — see section 6.

📊 5. Reading the primer pair table

Column	Meaning	Good value
Forward / Reverse primer	The two oligo sequences, 5′→3′. Amber = 5′ tail; blue = annealing region.	—
Len	Primer length in nt.	18–24
Tm	Melting temperature in the selected model.	57–63 °C
GC%	Guanine + cytosine content.	40–60 %
Amplicon	Product size (bp).	Target-dependent
ΔTm	Difference in Tm between forward and reverse.	≤ 3 °C
Cross-dimer	ΔG₃₇ of the forward + reverse heterodimer.	> −5 kcal/mol (green)
Position	Start–end coordinates of each primer in the template.	—

Ranking: pairs are sorted by a combined score. The top pair usually has near-optimal Tm, GC, amplicon length, and weak dimers. Still check the numbers — the score cannot know your lab.

🔗 6. Secondary structures

Structure	What it is	ΔG threshold	Action if bad
Hairpin	The primer folds back on itself into a stem-loop.	> −3 kcal/mol	Pick another candidate.
Self-dimer	Two copies of the primer anneal to each other.	> −6 kcal/mol	Pick another candidate.
Cross-dimer	Forward + reverse primers anneal to each other.	> −5 kcal/mol	Try the next-ranked pair.

Colours in the results table: green = safe, amber = borderline, red = strong — expect trouble.

🧪 7. TaqMan probe design

A TaqMan probe is an oligo labelled with a 5′ fluorophore and a 3′ quencher. When the probe is intact, fluorescence is quenched. During extension the polymerase's 5′→3′ exonuclease activity cleaves the probe, releasing the fluorophore — this is the real-time signal.

Property	Recommended
Length	18–27 nt
Tm	8–10 °C higher than the primers (66–72 °C)
GC content	40–60 %
5′ base	Must not be G (quenches the reporter)
Position	Between the primers, ideally near the forward primer
Homopolymer	No more than 4 consecutive same base

Tip: When designing a diagnostic assay, place the probe across an exon–exon junction so it cannot amplify genomic DNA.

🎯 8. Nested PCR

Nested PCR uses two primer pairs in consecutive reactions. The outer pair amplifies a larger region; the inner pair then re-amplifies a subset of that product. This dramatically increases specificity — useful when the template is rare, degraded, or contaminated with non-target DNA.

Reading the output

Outer pair — the large amplicon (usually 500–2000 bp). Run 15–20 cycles.
Inner pair — sits ≥ 40 bp inside the outer amplicon. Transfer 1 µL of the first reaction into a fresh master mix and run 25–30 cycles.
The tool automatically places the inner pair inside the top-ranked outer amplicon, with a 40-bp safety margin on each side.

Avoid contamination: use separate pipettes, fresh tips, and physically separated pre- and post-PCR areas. Nested PCR is extremely sensitive to carry-over.

🔬 9. Primer analysis mode

Paste existing primers (one per line, optionally name sequence) and the tool reports for each one:

Length, GC %, Tm (NN and Wallace), whole-primer ΔG₃₇, 3′-end ΔG.
Hairpin ΔG and self-dimer ΔG.
Number of G/C in the last five nucleotides (GC clamp).
Longest homopolymer run.

And for the set as a whole:

A cross-dimer matrix showing the ΔG for every primer pair. Red cells mean strong dimers — replace or redesign those primers.

🏷️ 10. 5′ tails and modifications

A 5′ tail is extra sequence added to the 5′ end of the primer. It does not anneal to the template but becomes part of the amplicon. Common uses:

Tail	Purpose
Kozak (GCCACC)	Enhances translation initiation in eukaryotic expression.
T7 / T3 / SP6 promoter	Adds a bacteriophage promoter for in vitro transcription.
M13 forward / reverse	Universal sequencing primer binding site.
EcoRI, BamHI, HindIII, …	Adds a restriction site for downstream cloning.
Custom	Any user-specified sequence — e.g. a homology arm for Gibson assembly.

Important: The tail is not included in the Tm or GC calculation, because it does not bind the template. In the results table, tails are highlighted in amber.

⚠️ 11. Common pitfalls & troubleshooting

No product (no amplification). Usually a too-strict Tm window, wrong annealing temperature, or a mismatch in the input sequence. Relax the stringency setting and try again; if the tool still fails, widen the Tm range.

Smear or multiple bands. Primers have a strong cross-dimer or self-dimer, or the annealing temperature is too low. Choose a pair with green cross-dimer and raise Ta by 2–3 °C.

Primer dimers on the gel. A bright band near 50 bp. Redesign with the analysis mode to see which primer pair is the culprit.

Genomic DNA contamination in RT-qPCR. Design at least one primer or the probe across an exon–exon junction. Also treat samples with DNase I before reverse transcription.

SNPs under the primer. A common SNP inside the annealing region can abolish amplification in some individuals. Check dbSNP for your target region.

Best practice: design 2–3 independent primer pairs per target, test them in parallel, and keep the one that gives a clean single band. Sequence the amplicon at least once to confirm it is the intended product.

Pre-order checklist

✔ 18–24 nt long
✔ GC between 40–60 %
✔ Tm 57–63 °C and ΔTm ≤ 3 °C
✔ Hairpin, self-dimer, cross-dimer all green
✔ No long homopolymer run
✔ Amplicon size appropriate for the application
✔ Checked against Primer-BLAST for specificity
✔ For expression: reading frame and Kozak verified
✔ 2–3 pairs tested in parallel

1 Mode & template

2 Target region

3 Parameters

i Notes on the calculations

📘 Tutorial & Result Interpretation

📑 On this page

🧬 1. Primer design basics

📏 2. The golden rules

🎯 3. Choosing the right mode

🌡️ 4. Reading Tm, GC and ΔG

Tm (melting temperature)

GC content

ΔG37 (Gibbs free energy)

📊 5. Reading the primer pair table

🔗 6. Secondary structures

🧪 7. TaqMan probe design

🎯 8. Nested PCR

Reading the output

🔬 9. Primer analysis mode

🏷️ 10. 5′ tails and modifications

⚠️ 11. Common pitfalls & troubleshooting

Pre-order checklist

ΔG₃₇ (Gibbs free energy)