pepkio-ligo-ratio-calc

Calculate exact insert-to-vector molar stoichiometry, required DNA masses, and reaction pipetting volumes for molecular cloning protocols.

What It Does

pepkio-ligo-ratio-calc simplifies DNA ligation reaction planning by determining the required insert mass (in nanograms) and stock solution volumes (in microliters) needed for target insert-to-vector molar ratios (e.g., 1:1, 3:1, 5:1, 10:1). It performs automated length-to-mass conversions, handles single or multi-insert assemblies, and validates whether required DNA volumes fit within total reaction limits.

Features

• Multi-Ratio Calculations: Computes required insert mass and pipetting volumes across multiple target molar ratios simultaneously.
• Volumetric Feasibility Validation: Checks reaction volume budgets (vector, insert, buffer, enzyme, water) and alerts when stock concentrations are insufficient.
• Flexible Unit Parsing: Accepts vector and insert lengths in base pairs (bp) or kilobases (kb), and DNA concentrations in ng/µL or µg/mL.
• Multi-Insert & Assembly Support: Accommodates cohesive-end, blunt-end, TA cloning, and multi-fragment assembly workflows.
• Python API & CLI: Provides a typed Python SDK (PepkioClient) and a command-line interface (pepkio-ligo-ratio-calc).

Installation

pip install pepkio-ligo-ratio-calc

Quick Example

import os
from pepkio_ligo_ratio_calc import PepkioClient

api_key = os.getenv("PEPKIO_API_KEY")

with PepkioClient(api_key=api_key) as client:
    result = client.run({
        "mode": "standard",
        "cloning_preset": "sticky_end",
        "vector_size": 3000,
        "vector_size_unit": "bp",
        "vector_concentration": 50,
        "vector_conc_unit": "ng_uL",
        "vector_mass_ng": 50,
        "reaction_volume_ul": 10,
        "buffer_volume_ul": 1,
        "enzyme_volume_ul": 1,
        "ratios": [1, 3, 5],
        "inserts": [
            {
                "id": "ins1",
                "name": "Target Gene Insert",
                "size": 1000,
                "size_unit": "bp",
                "concentration": 20,
                "conc_unit": "ng_uL"
            }
        ]
    })
    
    print("Run Status:", result.status)
    print("Pipetting Plan:", result.result)

Typical Use Cases

• Cohesive-End (Sticky-End) Subcloning: Determine insert mass for 3:1 or 5:1 molar ratios when ligating restriction digest products into linearized vectors.
• Blunt-End PCR Fragment Ligation: Scale insert stoichiometry (5:1 to 10:1) to optimize bimolecular collision rates for blunt-ended fragments.
• Multi-Fragment & Modular Assembly: Calculate balanced fragment masses for dual-insert ligations, Gibson Assembly, or Golden Gate cloning.
• Volume Constraint Validation: Verify if low-concentration DNA preparations can fit into standard 10 µL or 20 µL T4 ligase reactions without exceeding volume budgets.

Scientific Background

DNA ligation kinetics depend on relative molar end concentrations rather than raw mass concentrations. Molar amounts of double-stranded DNA are calculated from mass and fragment length using the average dsDNA molecular weight (~650 Da/bp):

$$\text{pmol dsDNA} = \frac{\text{Mass (ng)} \times 1000}{\text{Length (bp)} \times 650}$$

To achieve a target insert-to-vector molar ratio ($R$), the required insert mass is determined by:

$$\text{Insert Mass (ng)} = \text{Vector Mass (ng)} \times \left( \frac{\text{Insert Size (bp)}}{\text{Vector Size (bp)}} \right) \times R$$

Pipetting volume is calculated as $\text{Insert Mass (ng)} / \text{Insert Concentration (ng/\mu L)}$. Total volume is completed with nuclease-free water after accounting for fixed buffer, enzyme, and vector volumes.

Web Application

For researchers who prefer a graphical interface, an interactive web version is available.

Web Application: https://www.pepkio.com/tools/ligo-ratio-calc

The web interface features real-time volume budget visualization, cloning preset selection, shareable permalinks, and printable benchtop worksheets.

Documentation and Resources

GitHub Repository: https://github.com/pepkio/pepkio-ligo-ratio-calc

Web Application: https://www.pepkio.com/tools/ligo-ratio-calc

Source code and issue tracking are available on GitHub.

About Pepkio

Pepkio (https://www.pepkio.com/) develops software tools and bioinformatics solutions for life science researchers, including laboratory calculators and analysis services (https://www.pepkio.com/cro). See https://www.pepkio.com for additional tools and services.

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