Cloning

Molecular cloning – the isolation and multiplication of a defined DNA sequence – is a routine activity in molecular biology laboratories. Cloning typically involves PCR amplification of the DNA fragment of interest, followed by insertion into a suitable vector. The latter may be achieved in a variety of ways, the most common of which exploit (i) unique restriction sites present in the cloning vector and at the 5’- and 3’-ends of the PCR amplicon (insert), or (ii) dA-overhangs added to the 3’-ends of each DNA strand of the insert during PCR, which allow for cloning into linearized vectors with dT-overhangs. Both of these strategies require the use of a DNA ligase, to form covalent phosphodiester bonds between the 3’-hydroxyl and 5’-phosphate groups of terminal nucleotides of insert and plasmid DNA strands prior to transformation.

High fidelity amplification is required for many cloning applications in order to minimize spurious mutations introduced by the DNA polymerase during PCR. 

KAPA HiFi DNA Polymerase is an engineered Family-B polymerase that exhibits exceptional fidelity (100x lower error rate than Taq polymerase), sensitivity, and robustness.

KAPA HiFi DNA Polymerase is recommended for high fidelity PCR for inserts up to 15 kb. KAPA HiFi is compatible with blunt-end cloning.

KAPA Long Range DNA Polymerase is recommended for amplification from low copy number templates and is compatible with TA cloning. The error rate of KAPA Long Range is 3x – 6x lower than Taq polymerase.

KAPA T4 DNA Ligase is recommended for high efficiency cohesive and blunt-end ligations.

KAPA Rapid Ligase is recommended for fast (5 – 15 minutes at room temperature) and efficient cohesive and blunt-end ligations.