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Molecular biology Cloning

DNA Assembly Mix Calculator (Gibson / Golden Gate)

Calculate required DNA mass and volume from backbone/insert molar ratios for Gibson or Golden Gate. If concentrations are provided, volumes and remaining water for the total reaction are calculated.

All calculations run in your browser. No data is sent.

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How to use (3 steps)

  1. Select an example or enter fragment length (bp) and concentration (ng/µL) for backbone and inserts.
  2. Choose the molar ratio (e.g., inserts 2×) and the backbone amount (pmol or ng).
  3. The required mass (ng) and volume (µL), plus a recipe (master mix/reagents and remaining water), will appear.

This is a quantity calculator. Recommended conditions vary by kit and protocol, so follow your protocol. Golden Gate reagent presets are examples for calculation only.

Inputs (fragment table)

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Molar ratios reflect molecule counts. Different lengths mean different molecule counts at the same mass.
Details (molecular weight per bp)
Use 660 g/mol/bp as an approximate value for dsDNA.
Name Role Length (bp) Concentration (ng/µL) Actions
Paste TSV (optional)

Paste from Excel/Sheets (tab-separated).

Results

Remaining water (µL)
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Total DNA volume (µL)
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Total DNA mass (ng)
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2× master mix volume (µL)
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Fragment Role Length (bp) Target (pmol) Mass (ng) Volume (µL) Note

This tool calculates amounts only. It does not guarantee success.

How it’s calculated

  1. As a dsDNA approximation, pmol = (mass_ng × 1000) / (bp × 660).
  2. Set the backbone amount (pmol for Gibson, ng for Golden Gate) and compute backbone pmol.
  3. Each insert target is insert_target_pmol = backbone_pmol × ratio, then converted to mass (ng).
  4. If concentration (ng/µL) is known, volume = mass / conc gives volume (µL).
  5. Remaining water is calculated from total reaction volume minus master mix/reagents and DNA volume.

Values are guides. Follow your kit/protocol for final conditions.

FAQ

What is the difference between Gibson and Golden Gate?
Both assemble DNA fragments, but the reaction mechanisms and enzymes differ. This tool calculates how much DNA to mix.
Why use molar ratios?
Fragments of different lengths have different molecule counts at the same mass. Molar ratios keep molecule counts consistent.
What ratios are commonly used?
As a rule of thumb, inserts are often 2× or 3× the backbone. It depends on the assembly and should be tested.
Can I use this without concentration values?
Yes. You can still calculate required mass (ng). Volumes (µL) are shown only when concentrations are provided.
My volumes are extremely small (e.g., 0.1 µL).
Very small volumes are error-prone. Consider intermediate dilutions or adjust backbone amount and reaction volume.
Can I use the Golden Gate reagent presets as-is?
Reagent volumes depend on the kit and protocol. Presets are examples for calculation only; follow your protocol.
Will these amounts guarantee success?
No. This tool only calculates amounts. Success depends on design, DNA quality, and reaction conditions.