Ligation: Difference between revisions
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Revision as of 19:44, 17 February 2014
Ligation Protocol with T4 DNA Ligase
- Set up the following reaction in a microcentrifuge tube on ice.
- 2uL of 10X T4 DNA ligase Buffer
- 50 ng 50 ng (0.025 pmol) vector DNA (3 kb)
- 50 ng (0.076 pmol) insert DNA (1 kb)
- 1 uL of T4 DNA Ligase
- add nuclease-free water to make the total volume 20 uL
- Gently mix the reaction by pipetting up and down and microfuge briefly.
- For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
- For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours(alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation).
N.B (T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert.)
troubleshooting
- to avoid frequent thawing and freezing of the ligase buffer, prepare working stock of ligation buffer