• 1.0 μL 10X T4 ligase buffer
• 6:1 molar ratio of insert to vector
• We used 10 ng of vector.
• (8.5 - vector and insert volume) μl nuclease-free water
• 0.5 μL T4 Ligase

Calculating Insert Amount

• Insert Mass in ng = 6 x (Insert Length in bp/Vector Length in bp) x Vector Mass in ng
• The insert to vector molar ratio can have a significant effect on the outcome of a ligation and subsequent transformation step. Molar ratios can vary from a 1:1 insert to vector molar ratio to 10:1. It may be necessary to try several ratios in parallel for best results.
• If there is more than one insert (3A or Standard Assembly), calculate for the same amount of insert as you would if there were only one insert.

Procedure:

1. Add appropriate amount of deionized H2O to sterile microcentrifuge tube
2. Add 1 μL ligation buffer to the tube.
• Vortex buffer before pipetting to ensure that it is well-mixed.
• Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation.
3. Add appropriate amount of insert to the tube.
4. Add appropriate amount of vector to the tube.
5. Add 0.5 μL ligase.
• Vortex ligase before pipetting to ensure that it is well-mixed.
• Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 0.5 μL, just touch your tip to the surface of the liquid when pipetting.
6. Let the 10 μL solution sit at room temperature for 20 minutes.
7. Let the solution sit at 16°C for at least 2 hours.
8. Store at -20 degrees C.