Cloning method | QuickStep-Cloning | RF | ABI-REC | RAM | EMP | IFPC |
---|---|---|---|---|---|---|
Cloning strategy | Megaprimer | Megaprimer | Megaprimer | Megaprimer | Megaprimer | Megaprimer |
Amplification mode | Exponential | Linear | Exponential | Exponential | Exponential | Exponential |
Transformed product | Nicked-circular plasmid (2 nicks per plasmid) | Nicked-circular plasmid (2 nicks per plasmid) | Linear DNA | Linear DNA | Closed-circular plasmid | Closed-circular plasmid |
E. coli cells used | Chemically competent DH5α and C41 (DE3) | Electrocompetent TG1 | Chemically competent DH5α | Strain type not reported | Chemically competent DH5α | Chemically competent TOP10 |
In vivo homologous recombination | No | No | Yes | Yes | No | No |
Enzymatic phosphorylation-ligation | No | No | No | No | Yes | Yes |
Number of primers required | 4 | 2 | 3 | 3 | 3 | 3 |
Gel purification | No | No | No | 1× | No | Strongly recommended |
PCR purification | 1× | 1× | No | No | 2× | No |
Estimated cloning timea | 5 h 15 min | 14 h | 7 h 45 min | 7 h 45 min | 7 h 15 min | 6 h 30 min |
Reported cloning efficiencyb | 93–97 % | ~90 % c | 93–100 % | 75–94 % | 10–100 % | ~90 % |
Reference | - | [8] | [12] | [13] | [14] | [15] |