DNA ligation

Added By

• --BC 20:40, 26 Apr 2005 (EDT)

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