How to use (3 steps)
- Select an example or enter fragment length (bp) and concentration (ng/µL) for backbone and inserts.
- Choose the molar ratio (e.g., inserts 2×) and the backbone amount (pmol or ng).
- The required mass (ng) and volume (µL), plus a recipe (master mix/reagents and remaining water), will appear.
This is a quantity calculator. Reaction conditions still depend on your kit and protocol. Golden Gate reagent presets are examples for planning only.
Worked example
Suppose you are assembling a 3,000 bp backbone with one 900 bp insert at a 1:3 molar ratio. Set the backbone target first, then let the calculator convert that ratio into the insert mass and transfer volume. If one transfer is too small to pipette accurately, increase the backbone target or dilute the stock before running the reaction.
Inputs (fragment table)
Results
| 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
- As a dsDNA approximation, pmol = (mass_ng × 1000) / (bp × 660).
- Set the backbone amount (pmol for Gibson, ng for Golden Gate) and compute backbone pmol.
- Each insert target is insert_target_pmol = backbone_pmol × ratio, then converted to mass (ng).
- If concentration (ng/µL) is known, volume = mass / conc gives volume (µL).
- 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.
How to plan Gibson or Golden Gate assembly with this calculator
Use this page as a reaction-planning worksheet, not as a success guarantee. Pick the assembly method first, set one backbone baseline, and then calculate insert mass and volume from that ratio.
When to choose Gibson vs Golden Gate
Gibson is usually the better starting point when you have overlapping ends and want to reason in pmol across backbone and inserts. Golden Gate is more useful when your kit or protocol already defines the reaction mix and you mainly need to size DNA inputs around that setup.
How to set the backbone baseline
For Gibson, begin with the backbone amount in pmol and treat it as the anchor for every insert. For Golden Gate, begin with the backbone mass or the kit-style DNA baseline you actually pipette. Once the backbone is fixed, the insert targets become much easier to review.
How to read backbone:insert ratios
A ratio such as 1:2 or 1:3 is about molecule count, not raw mass. Longer fragments need more ng to reach the same pmol, so two inserts with the same ratio can still need very different masses. Plan the ratio before you decide the transfer volume.
Mass vs volume
Use mass (ng) to decide whether the molecular target is sensible. Use volume (µL) only after you trust the concentration values. If the mass looks right but the transfer volume is tiny, keep the molecular target and solve the pipetting problem with dilution or a larger DNA baseline.
Very small transfer volumes
Volumes around 0.1 µL to 0.3 µL are often too fragile for routine pipetting. If you see that pattern, make an intermediate dilution, increase the backbone amount, or choose a larger total reaction volume before you lock the protocol.
Worked example
Suppose you want a Gibson mix with one backbone and two inserts. Set the backbone to 0.02 pmol, then use a 1:2 ratio for each insert. The calculator holds the backbone constant, converts each insert target pmol into ng from fragment length, and then into µL from concentration. If one insert needs only 0.2 µL, keep the same pmol target and dilute that stock instead of changing the ratio blindly.
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FAQ
Can I use this as a Gibson assembly calculator?
Why use backbone and insert molar ratios?
What ratios are commonly used for Gibson assembly?
Can I use this without concentration values?
What if my calculated volumes are extremely small, such as 0.1 µL?
Can I use the Golden Gate reagent presets as-is?
Will these amounts guarantee success?
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