Length of the insert DNA fragment (e.g., 1500 bp or 1.5 kb)

Length of the linearized vector (e.g., 5000 bp or 5 kb)

Amount of vector DNA to use (typically 50-100 ng)

Molar ratio of insert to vector molecules

Results:

1. Ligation Insert Mass Formula

Calculate the mass of insert needed for desired molar ratio:

Where: All lengths must be in the same units (bp or kb), Molar ratio is insert:vector (e.g., 3 for 3:1 ratio)

Example: 50 ng vector (5 kb), 1.5 kb insert, 3:1 ratio → Insert = 50 × (1.5/5) × 3 = 45 ng

2. Molar Amount Calculation

Calculate moles of DNA from mass and length:

Where: 660 Da/bp = average molecular weight of double-stranded DNA base pair

This formula explains why equal masses of different-length fragments contain different numbers of molecules, necessitating molar ratio calculations.

3. DNA Concentration in Reaction

Calculate final DNA concentration for optimal ligation:

Optimal range: 1-10 µg/mL (0.001-0.01 µg/µL or 1-10 ng/µL)

Example: 100 ng total DNA in 20 µL → Concentration = 100/20 = 5 ng/µL ✓ Optimal

4. Multiple Insert Ligation

For cloning multiple inserts into one vector:

For 2 inserts at 6:1 total ratio: Each insert = Vector × (Insert length / Vector length) × 3. Use Gibson Assembly or NEBuilder for multiple inserts.

Step 1: Prepare DNA Fragments

Digest vector and insert DNA with appropriate restriction enzymes. Gel-purify fragments to remove enzymes and salts. Quantify DNA concentration using NanoDrop or spectrophotometry.

Step 2: Enter Fragment Lengths

Input the exact length of your insert and linearized vector in base pairs (bp) or kilobase pairs (kb). Accurate lengths are crucial for correct molar ratio calculations.

Step 3: Specify Vector Amount and Ratio

Enter the amount of vector you'll use (typically 50-100 ng). Select appropriate molar ratio: 3:1 for cohesive ends, 5-10:1 for blunt ends, or 1:1 for large inserts.

Step 4: Calculate and Set Up Reaction

Calculate insert mass needed. Set up ligation reaction with calculated amounts of insert and vector, ligase buffer, and T4 DNA ligase. Incubate according to protocol (typically 1 hour at room temperature or overnight at 16°C).

Example 1: Standard Cohesive End Ligation

Scenario: Cloning a 1.5 kb PCR product into a 5 kb plasmid vector

Given: Vector = 50 ng, Insert = 1.5 kb, Vector = 5 kb, Ratio = 3:1

Calculation:

Insert mass = 50 ng × (1.5 kb / 5 kb) × 3

Insert mass = 50 × 0.3 × 3

Insert mass = 45 ng

Use 45 ng of insert DNA with 50 ng vector for optimal 3:1 molar ratio.

Example 2: Blunt End Ligation

Scenario: Cloning blunt-ended 800 bp fragment into 4 kb vector

Given: Vector = 100 ng, Insert = 800 bp, Vector = 4000 bp, Ratio = 10:1 (blunt end)

Calculation:

Insert mass = 100 ng × (800 bp / 4000 bp) × 10

Insert mass = 100 × 0.2 × 10

Insert mass = 200 ng

Higher ratio compensates for lower blunt-end ligation efficiency.

Example 3: Large Insert Cloning

Scenario: Cloning 8 kb gene into 6 kb BAC vector

Given: Vector = 50 ng, Insert = 8 kb, Vector = 6 kb, Ratio = 1:1 (large insert)

Calculation:

Insert mass = 50 ng × (8 kb / 6 kb) × 1

Insert mass = 50 × 1.333 × 1

Insert mass = 67 ng

Equal molar ratio (1:1) preferred for large inserts to avoid multiple insertions.

Optimal Molar Ratios

Cohesive (Sticky) Ends: 3:1 to 5:1 insert:vector ratio provides best balance of efficiency and specificity.

Blunt Ends: 5:1 to 10:1 ratio compensates for 10-100× lower efficiency than cohesive ends.

Large Inserts (>5 kb): 1:1 ratio prevents multiple insertions and improves transformation efficiency.

Small Inserts (<500 bp): 5:1 to 10:1 ratio ensures sufficient insert molecules.

DNA Concentration

Total DNA: Keep between 1-10 µg/mL (1-10 ng/µL) in final reaction volume.

Vector Amount: Use 50-100 ng for standard reactions, can reduce to 10-25 ng to conserve materials.

High Concentration: May cause intermolecular ligation and background colonies.

Reaction Conditions

Temperature: Room temperature (25°C) for 10-60 min, or 16°C overnight for difficult ligations.

Ligase Amount: 400 units (1 µL of NEB T4 Ligase) for 20 µL reaction. Can reduce to 200 units.

PEG 4000: Included in most ligase buffers (5-15%) to increase effective DNA concentration.

What is DNA ligation?

DNA ligation is the process of joining two DNA fragments together by forming a phosphodiester bond between the 3'-hydroxyl end of one fragment and the 5'-phosphate end of another. T4 DNA ligase is commonly used to catalyze this reaction in molecular cloning.

How do you calculate insert to vector molar ratio?

Use the formula: Insert mass (ng) = Vector mass (ng) × (Insert length / Vector length) × Molar ratio. For a 3:1 insert:vector ratio with 50 ng of 3 kb vector and 1 kb insert: Insert mass = 50 × (1/3) × 3 = 50 ng.

What is the optimal insert to vector ratio for ligation?

For cohesive (sticky) end ligations, a 3:1 insert:vector molar ratio is optimal. For blunt end ligations, use 5:1 to 10:1 ratios due to lower efficiency. Large inserts (>5 kb) work better at 1:1 ratios.

Why is molar ratio important in ligation?

Molar ratio ensures the correct number of insert molecules relative to vector molecules, not just mass. Since DNA fragments of different lengths have different molecular weights, calculating molar ratios prevents using too much or too little insert.

What is the difference between sticky end and blunt end ligation?

Sticky (cohesive) end ligation joins DNA fragments with complementary overhangs, providing high efficiency and specificity (3:1 ratio recommended). Blunt end ligation joins fragments with no overhangs, requiring higher insert ratios (5:1 to 10:1) and often PEG for efficiency.

How much vector DNA should I use in a ligation?

Standard ligation reactions use 50-100 ng of vector DNA. Some protocols use as little as 10-25 ng to conserve materials. Keep total DNA concentration between 1-10 µg/mL (0.1-1 ng/µL) in the final reaction volume.

Why do I get empty vector colonies after ligation?

High background is usually due to incomplete vector digestion or lack of dephosphorylation. Treat vector with Antarctic Phosphatase (CIP) or gel-purify to remove uncut vector. Ensure complete digestion by checking on gel before ligation.

Can I use this calculator for Gibson Assembly?

Yes, but Gibson Assembly typically uses 2-3× molar excess of inserts (2:1 to 3:1 ratio). Use NEBuilder Assembly Tool for multiple inserts. Gibson is more forgiving than restriction ligation and works with a wider range of ratios.

Problem: No Colonies

Causes: Inactive ligase, improper vector dephosphorylation, incompatible ends, damaged DNA

Solutions: Check ligase activity with positive control, ensure fresh ATP in buffer, verify compatible ends, use freshly digested DNA, check vector:insert ratio (try 1:1, 3:1, 5:1)

Problem: High Background (Empty Vector)

Causes: Incomplete digestion, lack of dephosphorylation, self-ligation

Solutions: Treat vector with CIP/Antarctic Phosphatase, gel-purify vector, increase digestion time, verify complete digestion on gel

Problem: Wrong Insert Orientation

Causes: Using same enzyme for both ends, non-directional cloning

Solutions: Use two different restriction enzymes for directional cloning, screen colonies by PCR or restriction digest to identify correct orientation

Problem: Multiple Inserts

Causes: Excess insert DNA, high DNA concentration, blunt end ligation

Solutions: Reduce insert:vector ratio to 1:1 or 2:1, dilute DNA to 1-5 ng/µL, use cohesive ends instead of blunt ends