DNA ligation
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Ligation Mix
- Example - 10ul mix
- 1.0 $\mu$L 10X T4 ligase buffer
- 0.5 $\mu$L T4 Ligase
- 6:1 Molar ratio of insert to vector ($\sim$10ng vector)
- X$\mu$l ddH$_2$O to bring the total to 10$\mu$l
Calculating Insert and Vector Amounts
[math]\displaystyle{ Insert Mass = 6\times\left[\frac{Insert Length}{Vector Length}\right]\times Vector Mass }[/math]
Reaction Conditions
- Let the 10 $\mu$l solution sit at 22.5$^o$C for 30min
- Denature the ligase at 65$^o$C for 10min
- Dialyze for 20 minutes if electroporating
- Use disks shiny side up
- Store at -20C