Corum:T4 Ligation: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Sean P Corum (talk | contribs) m (→Procedure) |
Sean P Corum (talk | contribs) m (→Materials) |
||
| Line 9: | Line 9: | ||
For a 10 μL ligation reaction: | For a 10 μL ligation reaction: | ||
* 5 μL linker dsDNA (either | * 5 μL ~100nM linker dsDNA (either hybridization product or digested, purified PCR product) | ||
* 0.5 μL backbone dsDNA ( | * 0.5 μL ~10nM backbone dsDNA (gel extracted digestion product from donor plasmid) | ||
* 2 μL sterile ddH<sub>2</sub>O | * 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 | ||
* 0.5 μL T4 ligase | * 0.5 μL T4 ligase | ||
(Note: standard ratio of backbone to linker given here is ~1:100. This may need to be adjusted for non-standard conditions.) | |||
==Procedure== | ==Procedure== | ||
Revision as of 10:38, 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 ~100nM linker dsDNA (either hybridization product or digested, purified PCR product)
- 0.5 μL ~10nM backbone dsDNA (gel extracted digestion product from donor plasmid)
- 2 μL sterile ddH2O
- 1 μL 10X ligation buffer
- 1 μL 10mg/ml BSA
- 0.5 μL T4 ligase
(Note: standard ratio of backbone to linker given here is ~1:100. This may need to be adjusted for non-standard conditions.)
Procedure
- 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.
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
References
Contact
or instead, discuss this protocol.
