- 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.
- Incubate 16 °C overnight (8 hr minimum).
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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