Corum:T4 Ligation: Difference between revisions
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For a 10 μL ligation reaction: | 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: ratio of backbone to linker should be | * 0.5 μL backbone dsDNA (usually ~10 nM) (Note: ratio of backbone to linker should be 1:100. The specific amounts of linker and backbone dsDNAs may need to be adjusted to keep this ratio for non-standard concentrations) | ||
* 2 μL sterile ddH<sub>2</sub>O | |||
* 1 μL 10X ligation buffer | * 1 μL 10X ligation buffer | ||
* 1 μL 10mg/ml BSA | * 1 μL 10mg/ml BSA |
Revision as of 08:35, 29 June 2012
- SC 15:16, 19 June 2012 (EDT):
Overview
Standard DNA ligation using NEB T4 ligase to form a vector.
Materials
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: ratio of backbone to linker should be 1:100. The specific amounts of linker and backbone dsDNAs may need to be adjusted to keep this ratio for non-standard concentrations)
- 2 μL sterile ddH2O
- 1 μL 10X ligation buffer
- 1 μL 10mg/ml BSA
- 0.5 μL T4 ligase
Procedure
- Add the reaction components together in order listed above. Mix gently and spin.
- Incubate 16 °C overnight (8 hr minimum).
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
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- Anecdotal observations that might be of use to others can also be posted here.
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References
Relevant papers, books, and websites
Contact
or instead, discuss this protocol.