Advanced Molar Ratio NEB Calculator
Generated July 21, 2026, 11:50 pm
Common molar-ratio comparison
The table compares insert requirements using current vector settings.
| Insert : Vector | Required insert mass | Insert amount | Estimated insert volume |
|---|---|---|---|
| 1 : 1 | 12.5 ng | 0.019 pmol | 0.625 µL |
| 2 : 1 | 25 ng | 0.038 pmol | 1.25 µL |
| 3 : 1 | 37.5 ng | 0.057 pmol | 1.875 µL |
| 5 : 1 | 62.5 ng | 0.095 pmol | 3.125 µL |
| 7 : 1 | 87.5 ng | 0.133 pmol | 4.375 µL |
| 10 : 1 | 125 ng | 0.189 pmol | 6.25 µL |
| 20 : 1 | 250 ng | 0.379 pmol | 12.5 µL |
Formula used
The calculator normalizes units before applying molecular relationships.
Required insert mass
Insert mass = Vector mass × Insert length ÷ Vector length × Ratio.
This preserves molecule counts across fragments with different lengths.
Mass to picomoles
pmol = ng × 1000 ÷ Fragment molecular weight.
Double-stranded DNA uses approximately 660 g/mol per base pair.
Stock volume
Volume = Required mass ÷ Stock concentration.
Both values are converted into nanograms and microliters first.
Batch scaling
Batch amount = Per-reaction amount × Reaction count × Overage factor.
The overage factor equals one plus the selected percentage.
Actual ratio
Insert:Vector = Insert picomoles ÷ Vector picomoles.
This reveals the molecular ratio created by known masses.
Multi-insert equivalent
Insert pmol = Vector pmol × Assigned molar equivalent.
Each insert mass then depends on its own fragment length.
How to use this calculator
Follow these steps for a complete ligation planning worksheet.
- Select the calculation mode matching your unknown quantity.
- Choose the nucleic-acid type used by the fragments.
- Enter vector and insert lengths with their units.
- Provide masses when the selected mode requires them.
- Enter stock concentrations to calculate pipetting volumes.
- Confirm whether the ratio reads insert-to-vector or vector-to-insert.
- Choose a ratio preset or enter a custom ratio.
- Open multi-insert mode when planning several fragments.
- Set reaction count, overage, buffer, ligase, and additives.
- Review warnings before preparing the final reaction mixture.
- Export CSV or JSON records for laboratory documentation.
- Use Print / PDF to save the complete worksheet.
Understanding molar ratios
Mass ratios and molecular ratios describe different experimental quantities.
Why fragment length matters
A longer DNA fragment has a greater molecular weight than a shorter fragment. Equal nanogram quantities therefore contain different numbers of molecules. Molar-ratio planning corrects that difference before ligation.
Mass ratio versus molar ratio
A mass ratio compares measured DNA weights directly. A molar ratio compares molecule counts after accounting for fragment length. Ligation planning usually needs the second relationship.
Ratio orientation
A displayed ratio of 3:1 means different things under different orientations. Insert:Vector 3:1 means three insert molecules per vector molecule. Vector:Insert 3:1 represents the inverse relationship.
Pipetting realism
Very small calculated volumes may be difficult to pipette accurately. This calculator compares exact volumes against a configurable minimum. It then suggests dilution when volumes fall below that threshold.
Experimental responsibility
Calculated quantities are planning estimates rather than performance guarantees. Fragment purity, end compatibility, phosphorylation, enzyme condition, and buffer quality matter. Confirm every protocol against current manufacturer instructions and laboratory standards.
Frequently asked questions
These answers explain common planning decisions and limitations.
What does a 3:1 ratio mean?
Under Insert:Vector orientation, it means three insert molecules per vector molecule.
Why does the insert mass change with length?
Longer fragments need more mass to provide the same molecule count.
Can the calculator handle several inserts?
Yes. Multi-insert mode calculates each fragment from its assigned equivalent.
Why is my stock volume zero?
The corresponding concentration is missing, zero, or unsupported.
What should I do with tiny volumes?
Dilute the stock so the required mass uses a practical volume.
Does circular DNA change the mass equation?
Topology does not substantially change the basic molecular-weight estimate used here.
Can I use RNA values?
Yes. Select RNA to use the approximate nucleotide molecular-weight factor.
How is overage applied?
Every batch component is multiplied by reaction count and the overage factor.
Can I save the setup?
Yes. The local preset buttons store or reload the form in your browser.
Does Print / PDF create a PDF server-side?
It opens the browser print dialog, where PDF saving is usually available.
Is this a replacement for a validated protocol?
No. It is a planning tool and must be checked against current procedures.
Can ratio orientation be reversed?
Yes. The calculator normalizes either orientation into insert-to-vector form.