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Self ligation
1. Dilution of DNA favors self-ligation.
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Linear DNA ( 10-100 ng)
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1 ml
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10X ligation buffer
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10 ml
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T4 ligase (5 units)
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1 ml
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ddH2O
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88 ml
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Insert into a vector
1a.
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Vector DNA ( 20-200 ng)
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1 ml
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Insert DNA ( 60-600 ng) ( 3 times vector)
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1 ml
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10X ligation buffer
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2 ml
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T4 ligase (5 units)
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1 ml
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ddH2O
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15 ml
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2. Incubate the ligation mixture at 16 °C for 2-3 hours or overnight.
3. Inactivate T4 DNA ligase by heating to 65 °C for 10 minutes or use directly
for transformation.
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