Corum:T4 Ligation: Difference between revisions
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Sean P Corum (talk | contribs) m (→Materials) |
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# Add the reaction components together in order listed above. Mix gently and spin. | # Add the reaction components together in order listed above. Mix gently and spin. | ||
# Incubate 16 °C overnight (8 hr minimum). | # Incubate 16 °C overnight (8 hr minimum). | ||
# [http://openwetware.org/wiki/Corum:Transformation Transform] 2.5 μL (or more, if necessary, to obtain an acceptable amount of colonies, for example) into chemically competent cells. | |||
==Notes== | ==Notes== |
Revision as of 10:30, 26 July 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: 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
Procedure
- Add the reaction components together in order listed above. Mix gently and spin.
- Incubate 16 °C overnight (8 hr minimum).
- Transform 2.5 μL (or more, if necessary, to obtain an acceptable amount of colonies, for example) into chemically competent cells.
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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References
Relevant papers, books, and websites
Contact
or instead, discuss this protocol.