- SC 15:16, 19 June 2012 (EDT):
Standard DNA ligation using NEB T4 ligase to form a vector.
For a 10 μL ligation reaction:
- 5 μL linker dsDNA (either 100nM standard concentration linker hybridization product or standard digested and purified PCR product, which is usually ~100nM)
- 0.5 μL backbone dsDNA (usually ~10 nM) (Note: standard ratio of backbone to linker is ~1:100. This may need to be adjusted for non-standard conditions.)
- 2 μL sterile ddH2O
- 1 μL 10X ligation buffer
- 1 μL 10mg/ml BSA
- 0.5 μL T4 ligase
- Add the reaction components together in order listed above. Mix gently and spin.
- Include -linker control reaction (sub sterile ddH2O for linker).
- Incubate 16 °C overnight (8 hr minimum).
- Transform 2.5 μL rea(or more, if necessary, to obtain an acceptable amount of colonies) into chemically competent cells.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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