Ligation: Difference between revisions
From Powers Wiki
(Created page with "==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) vecto...") |
No edit summary |
||
| (2 intermediate revisions by 2 users not shown) | |||
| Line 1: | Line 1: | ||
[[Category:Protein expression]] | |||
[[category:Cell_Culturing]] | |||
==Ligation Protocol with T4 DNA Ligase== | ==Ligation Protocol with T4 DNA Ligase== | ||
Latest revision as of 06:42, 20 January 2022
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