Ligation of Insert to pGEX DNA

Ready-To-Go T4 DNA Ligase can be used to achieve ligations in 30 to 45 min. An alternate procedure is described below.

Reagents Required

Linearized pGEX DNA Insert DNA ATP, 100 mM 10× One-Phor-All Buffer PLUS (OPA+): T4 DNA ligase

100 mM Tris acetate, 100 mM magnesium acetate, 500 mM potassium acetate, pH 7.5

Procedure

1. Prepare linearized pGEX DNA and insert DNA so that they will be present at a vector to insert ratio of 1:5 moles of ends. The moles of ends of linear DNA can be calculated with the following formula:
   moles of ends = 2 × (g of DNA)/[(# of bp) × (649 Daltons/bp)]
   Example: 100 ng of pGEX DNA (0.06 pmol of ends) would require 100 ng of a 1 kb insert (0.3 pmol of ends).
   - For ligation of cohesive ends, the final reaction mix should contain 1 mM ATP (diluted) and 0.5 to 5 units of T4 DNA ligase, and should be incubated for 1 to 4 h at 10 °C
   - For ligation of blunt ends, the final reaction mix should contain 0.1 to 1 mM ATP (diluted) and 10 to 15 units of T4 DNA ligase, and should be incubated for 2 to 16 h at 4 °C to 16 °C.
   
   
2. Based upon the above considerations in step 1, prepare the following reaction mixture specific for your application:
   1 to 5 µl of linearized pGEX DNA
   1 to 5 µL of insert DNA
   2 µL of 10× One-Phor-All Buffer PLUS (OPA⁺)
   0.2 µL of 100 mM ATP
   0.5 to 15 units of T4 DNA ligase
   Water to 20 µL
   
   
3. Incubate for either 1 to 4 h at 10 °C (cohesive ends) or 2 to 16 h at 4 °C to 16 °C (blunt ends).
   
   
4. Terminate the reaction by heating at 65 °C for 10 min.

The ligation reaction can be used directly to transform competent cells. Otherwise, it can be stored at -20 °C until needed.