Endy:DNA ligation using T4 DNA ligase

Ligation Mix

Example - 10ul mix

• 1.0 μL 10X T4 ligase buffer
  • Make sure this buffer is completely melted and dissolved. Precipitate is DTT. Probably best to aliquot this buffer into smaller portions, to reduce the freeze/thaw cycles. In general, make sure the buffer still smells strongly like "wet dog" (Checking if the DTT is still good.)
• 0.5 μL T4 Ligase
• 6:1 Molar ratio of insert to vector (~10ng vector)

• Dilute with ddH₂O to bring the total to 10μL

Calculating Insert and Vector Amounts

[math]\displaystyle{ \rm{Insert\ Mass} = 6\times\left[\frac{\rm{Insert\ Length}}{\rm{Vector\ Length}}\right]\times \rm{Vector\ Mass} }[/math]

Procedure

• Let the 10 μL solution sit at 22.5°C for 30 mins
• Denature the ligase at 65°C for 10min
• Dialyze for 20 minutes if electroporating
• Use disks shiny side up
• Store at -20°C