Ligation Calculator for Insert:Vector Ratio and Volumes
What this ligation calculator does
This calculator helps you plan a DNA ligation reaction by computing the required insert mass for a chosen insert:vector molar ratio, and converting masses into pipetting volumes using your stock concentrations. It also estimates buffer volume (10X to 1X final) and how much water you need to reach your final reaction volume.
Why molar ratio matters
Ligation efficiency depends strongly on how many DNA molecules (not just ng) are available to collide and ligate. Since longer fragments weigh more per molecule, a 3:1 ratio by mass is not the same as a 3:1 ratio by molecules. This is why molar ratio planning is standard for cloning workflows.
Core formula used
For double-stranded DNA, the molecular weight scales with length, approximately \(660\ \text{g/mol per bp}\). When you take a ratio, the constant cancels out and you can use a simple length-based scaling:
\[ m_{insert} = m_{vector}\times\left(\frac{bp_{insert}}{bp_{vector}}\right)\times R \]
Where:
- \(m_{insert}\) - required insert mass (ng)
- \(m_{vector}\) - chosen vector mass (ng)
- \(bp_{insert}\) - insert length (bp)
- \(bp_{vector}\) - vector length (bp)
- \(R\) - target molar ratio (Insert:Vector)
Converting ng to pmol
If you want the molar amount directly, the calculator uses:
\[ \text{pmol}=\frac{m_{ng}}{0.66\times bp} \]
This comes from \(\text{pmol}=\frac{m(g)}{MW(g/mol)}\times 10^{12}\) with \(MW \approx 660\times bp\).
Reaction volume planning
Once masses are known, volumes are computed with:
\[ V(\mu L)=\frac{m(ng)}{C(ng/\mu L)} \]
Ligase buffer is assumed to be 10X, so the volume for 1X final is:
\[ V_{buffer}=0.10\times V_{total} \]
Finally, water is the remainder:
\[ V_{water}=V_{total}-V_{vector}-V_{insert}-V_{buffer}-V_{ligase} \]
Practical guidance
Typical starting ratios
Common starting points are approximately 3:1 for sticky ends and 5:1 for blunt ends. These are starting points, not strict rules. If background is high, you may adjust ratios or vector preparation.
Dephosphorylation note
If your vector is not dephosphorylated, self-ligation risk is higher. This does not change the mass math, but it changes your expected background and troubleshooting priorities.
Pipetting accuracy
If the insert volume is extremely low (for example under \(0.2\ \mu L\)), consider diluting your insert stock so you can pipette more accurately while keeping the same total mass.
How to use the results table
The table provides a pipetting plan (vector, insert, buffer, ligase, water) and highlights critical conditions such as negative water volume, indicating the total volume is overfilled. The “Customize Columns” control lets you reorder and hide columns without breaking exports or table integrity.