Cloning Calculator

🧬 MOLECULAR CLONING SUITE

NEBuilder® HiFi • In-Fusion™ • Ligation Ratio Calculators

🔧 Gibson-Assembly/NEBuilder® HiFi

💉 In-Fusion™

🔗 Ligation Ratio

🔧 NEBuilder® HiFi Assembly Calculator

📋 DNA Fragments 1

Name	Length (bp)	Conc (ng/µl)	Type
			Vector Insert

📏 Tips: 2-3 frags: 0.05 pmol each • 4-6 frags: 0.2 pmol total

🧪 20 µl Reaction

Component	Volume	pmols
TOTAL	20 µl	0.000 pmol

⏱️ 15 min at 50°C 🧊 Store on ice

pmol = (ng × 1000) / (bp × 660) • Target: 0.05 pmol/fragment (≤3 frags)

💉 In-Fusion™ Assembly Calculator

Vector Size (bp)

Number of Inserts 1 2 3 4 5

📋 Insert Sizes (bp)

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

📊 DNA Amounts

Vector: 0 ng

Total DNA: 200 ng

Vector Molarity: 0 fmol

🧪 10µl Reaction

Vector 0 ng 0 µl

5X Mix 2 µl fixed

Water 8 µl 0 µl

⏱️ 15 min at 50°C

Vector: 200ng total • Insert: Vector × (InsertSize/VectorSize) × 2 • 5X mix: 2µl fixed

🔗 Ligation Ratio Calculator

Insert DNA Length

bp kb

Vector DNA Length

bp kb

Vector DNA Mass

ng µg pg

📏 1:1 = sticky ends 🔗 3:1 = blunt ends 🧪 5:1 = difficult inserts

📊 Required Insert Mass by Ratio

Formula: Insert Mass = Ratio × Vector Mass × (Insert Length / Vector Length)

Common ratios: 1:1 (cohesive), 3:1 (blunt), 5:1 (problematic)

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