Cloning Short Double-Stranded Inserts via T4 Ligation

Cloning short double-stranded inserts ~50-100bp in length into an expression vector can sometimes be tricky. The following is a summary of key parts of a method I have used and found to work effectively.

The Insert

For short inserts, I would have the insert synthesized as I do with regular PCR primers. Have your desired restriction sites added to the 5’ and 3’ ends. In addition to the restriction sites, I would also include a short stretch of ~8bp of random sequences to the ends of the restriction sites to serve as a cap. This is because restriction enzymes require a few bases on both sides of the restriction site in order to cut.

Order the inserts as you would with primers. As such, you will need a forward and reverse complement. Further, I go with the desalt purification.

Forming The Double-Stranded Insert

Your insert should arrive as two single strands so you will need to create the double-stranded insert for cloning. A simple way to do this is to reconstitute the individual tube and add an equal amount of both strands into a new tube.  Seal up the tube very tightly and have the tube float in a beaker of boiling water for 2 minutes. Turn off the heat and let the beaker cool at room temperature. By the next day your beaker of water should be at room temperature. Take the tube out of the water and give it a quick pulse spin. You now have double-stranded insert ready for restriction endonuclease digestion.

Restriction Enzyme Digestion

Once your restriction digest is complete, you should purify the insert to remove the small cleaved products. Your vector should also be digested with the same restriction enzymes and purified. An important step to do is to dephosphorylate the linearized vector. Use can do this using calf intestinal phosphatase.

Ligation

Use a standard ligation protocol. I generally use a ratio of 1(vector):3(insert) and leave the ligation reaction at 4 degrees overnight.

Transform as you normally do.