IGEM:Harvard/2007/Protocols/Ligation Protocol


 * 1) Add appropriate amount of deionized H2O to sterile 0.6 mL tube
 * 2) Add 1 &mu;L 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 &mu;L 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 1 &mu;L, just touch your tip to the surface of the liquid when pipetting.
 * 6) Let the 10 &mu;L solution sit at 22.5&deg;C for 30 mins
 * 7) Denature the ligase at 65&deg;C for 10min
 * 8) Dialyze for 20 minutes if electroporating
 * 9) Use disks shiny side up
 * 10) Store at -20&deg;C