Molecular cloning is a technique that many researchers will perform at least once during their career. These days it can be a fairly straight-forward procedure, especially with the large number of reagents that are available to help speed the process up. One of the longest steps (in terms of time) in the cloning process is the ligase step. DNA ligase is the modifying enzyme used to join both cohesive and blunt ended DNA to create recombinant DNA. This enzyme creates phosphodiester bonds between 3’hydroxyl group of one nucleotide with the 5’phosphate group of another nucleotide. The catalytic activity of this enzyme requires both ATP and Mg++ and the ligation reaction usually occurs within 1-3 hours at room temperature or can be completed in 14 hours at 4ºC.
However, if you don’t have a few extra hours to waste while your DNA is ligating, you may want to try New England BioLabs Quick Ligation Kit, which can create ligated DNA within 5 minutes at room temperature. I have used this kit while cloning many different DNA constructs. It is a convenient tool to have in any molecular biology laboratory as it is fast, it takes under 10 minutes to set up the reaction and allow the ligation to proceed. I have been able to ligate, transform and select for clones in ½ the time it would take if I used traditional ligation kits.
The kit comes with enough Quick enzyme and buffer to perform 30 different ligation reactions. All you need is 50ng of your vector and 3-fold molar excess of your insert diluted in 10ul of water. Next you add the 10ul of the 2X Quick ligation buffer and 1ul of the Quick T4 DNA Ligase. After mixing and a brief centrifuge, the samples incubate at room temperature for 5 minutes and can immediately be used in a transformation.
If I had to choose one product that I found not only to be reliable but to be a huge timesaver, it would have to be New England BioLabs Quick Ligation Kit. I have already recommended this item to my fellow labmates for its effectiveness and efficiency.
Sharon Yavrom
Graduate Student
University of Southern California