Ligation Calculator
Calculate optimal insert:vector ratios for molecular cloning
Note: This calculator determines optimal insert:vector ratios and ligation conditions for molecular cloning experiments.
Optimal Insert:Vector Ratio
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Insert Volume Required (μL)
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Total Reaction Volume (μL)
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Vector Amount (ng)
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Insert Amount (ng)
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Ligation Protocol
Enter parameters to see ligation protocol.
Understanding Ligation Ratios
- 3:1 Ratio: Standard ratio for most cloning applications
- 5:1 Ratio: Higher insert ratio for difficult ligations
- 1:1 Ratio: Equal molar ratio for specific applications
- Molar Ratio: Based on molecular weights of vector and insert
Ligation Best Practices
- Use fresh T4 DNA ligase and buffer
- Incubate at 16°C for 1-4 hours or overnight
- Heat inactivate ligase at 65°C for 10 minutes
- Transform competent cells immediately after ligation
- Use appropriate controls (vector only, insert only)
Important: This calculator provides theoretical values. Actual ligation efficiency
may vary due to insert/vector quality, ligase activity, and experimental conditions.
Always validate with appropriate controls.