Ligation Calculator

The Pipette Solution

HiFi Assembly

In-Fusion

Ligation Ratio

NEBuilder® HiFi Assembly

Final Volume

Fragments 1

Name	bp	ng/µl	Type
			VectorInsert

Reaction Setup

Component	Amount	Vol	Calc
TOTAL		20 µl

15 min at 50°C

pmol = (ng × 1000) / (bp × 660) · Master Mix = 50% of final volume

In-Fusion™ Assembly

Final Volume

Vector Size (bp)

Number of Inserts 1 2 3 4 5

Insert Sizes (bp)

Small fragments (<50 bp) may need 3:1 ratio

Reaction Setup

Component	Amount	Vol	Calc
TOTAL		10 µl

15 min at 50°C

Vector = 200 ng × scale / sumRatio · Insert = 2 × Vector × (Insert/Vector size) · 5X mix = Vol ÷ 5

Ligation Ratio Calculator

Final Volume

Insert DNA Length

bpkb

Vector DNA Length

bpkb

Vector DNA Mass

ngµg

Vector Stock (ng/µl)

Insert Stock (ng/µl)

Insert : Vector Ratio 1 : 1 (Cohesive) 2 : 1 3 : 1 (Blunt) 5 : 1 (Difficult) 7 : 1

Required Insert Mass by Ratio

Reaction Setup — Selected Ratio

Component	Amount	Vol	Calc
TOTAL		20 µl

1 hr at 16°C 65°C — 10 min inactivation

Insert Mass = Ratio × Vector Mass × (Insert ÷ Vector Length) · Buffer = Vol ÷ 10 · Ligase = Vol ÷ 20

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