2x Rapid Ligation Lab Report

Ligation is the process of joining two DNA fragments or other molecules by a phosphate ester linkage with the action of enzyme. Ligation of DNA is process that involve the DNA of interest is inserted into the plasmid. This 3’-hydroxyl of DNA terminus are joined together with 5’-phosphoryl of another by phosphodiester bond. The ligation of DNA fragments usually performed by using T4 DNA ligase. All the reaction components such as ligation buffer, DNA insert, pGEM®-T, and T4 DNA ligase is mixed by gentle pipetting. The 2X rapid ligation buffer is used in this experiment to saving the ligation time. This ligation buffer contains ATP. ATP is used to catalyze the formation of phosphodiester bond and the reaction of restriction enzyme buffer. The recommended time and temperature of incubation when using 2X rapid ligation buffer are one hour at room temperature (24°C) and overnight at 4°C. In this overnight incubation at 4°C is applied to achieve maximum number of recombinants.
The single 3’-T overhangs which usually called as “sticky ends” at the insertion sites helps to increase the ligation of DNA fragments into plasmid by preventing recircularization of vector. When the overhang is compatible with the base pairs, the two fragment of DNA will connect by ligation reaction. The new pGEM®-T and pGEM®-T Easy Vector Systems already contain the 2X rapid ligation buffer so that T4 DNA ligase can be used for one-hour ligation reactions instead of overnight ligation. T4 DNA ligase is used to catalyze the joining between 5’ phosphate and 3’ hydroxyl groups of adjacent nucleotides. The DNA ends can be cohesive ends or blunt ends configuration. Ligase usually performed at 4°C overnight or one hour at room temperature