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Ligation of Linearized Plasmid Vector and DNA Fragment with Sticky Ends
Materials
- Linearized and purified plasmid DNA
- Purified DNA fragment cut with the same restriction enzymes and proper orientation
- T4 ligase (Promega Cat# M1801, 3 U/m l)
- 10X Ligase buffer (Promega Cat# M1801)
- Nuclease-free water
- Agarose gel and apparatus and solution
- Gel scanner and software for densitometry
- 15oC water bath or ThermoMixer R
Procedure
- Check the vector and DNA fragment(s) concentration by running a agarose gel. Concentrate or dilute to proper concentration if necessary.
- Keep all reagents on ice. Add reaction components as follows:
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condition
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H2O
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Vector DNA
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Insert DNA(s)
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10X Buffer
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Ligase
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Vector only (background)
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Add to final 10ul
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100ng
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-
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1ul
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-
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Vector + Ligase (Control)
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Add to final 10ul
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100ng
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-
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1ul
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1ul
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Ratio I (1:1)
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Add to final 10ul
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100ng
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100 x (bpfrag/bpvector) ng
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1ul
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1ul
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Ratio II (1:3)
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Add to final 10ul
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100ng
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300 x (bpfrag/bpvector) ng
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1ul
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1ul
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Ratio III (1:5)
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Add to final 10ul
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100ng
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500 x (bpfrag/bpvector) ng
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1ul
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1ul
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- Mix and spin briefly. Incubate at 15oC for 16 hours.
- Use 5ul ligation reaction for transformation. The left 5ul can be used for ligation estimation by agarose gel.
- Use uncut vector as transformation effeciency control.
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