Part:BBa_K4235002:Design

Cloning technique

Ligation independent cloning is a technique alternative to traditional restriction enzyme cloning, which involves producing short homology arms/overhangs on the cloning substrates using PCR amplification. Vectors can be linearized using PCR primers or a restriction enzyme. For cloning into the pFastBac transfer vector, we used two primers (registry sequences) for linearizing the plasmid, which are attached to the 3’ and 5’ ends of the vector. Complementary primer sequences are used to amplify the insert for the cloning reaction. LIC exploits the dual polymerase - exonuclease activity of the T4 polymerase. The insert is treated with just dATPs and T4 polymerase, which chews back the 3’ ends until it reaches a T. Upon reaching a 3’ T, T4 polymerase gets stalled and switches its action to polymerase as it starts constantly adding dATP. Similarly, the vector is treated with just dTTPs and T4 polymerase. This treatment step produces DNA constructs with 5’ overhangs that can anneal to one another in the final annealing reaction.

Primer design for LIC:

The following primers were used to PCR amplify the insert and the vector and to add the extra sequence designed for this particular LIC reaction.

Insert 5’ : CCAGCGGCGGT GCCACCATGAGGGTC Insert 3’ : GAGCCCGAGGAGCT AGAATTCTTTGTCTTTTTCCAAAC

Vector 5’ : CCGCCGCTGGA GGTTTCGGACCGAGATC Vector 3’ : GCTCCTCGGGCTCA GGTACCGATTACGATATCCC