Successful DNA cloning is critical for DNA engineering workflows in medical, veterinary, and agricultural research. A single error in the finished construct could ruin downstream steps, and therefore, the initial screening process must locate correct clones. However, the process is costly and labor-intensive, especially when the cloning technique is inefficient and the resulting constructs are inaccurate.
In-Fusion Snap cloning offers distinct advantages over other commercially available seamless cloning kits, including various Gibson assembly-based methods (Table 1).
| In-Fusion Snap cloning | Gibson assembly-based cloning | |
|---|---|---|
|
Colony counts |
More, especially in difficult cloning experiments | Fewer or no colonies in difficult cloning experiments |
|
Polymerase-mediated fill in at junctions |
None: relies on competent cell machinery, leaving less vulnerability to error | Uses DNA polymerase: more prone to sequence errors at junctions |
|
Incubation time |
15 minutes | Up to 60 minutes |
|
Ligase-mediated sealing in vitro |
No ligase: fewer chances of empty vectors to re-ligate, yielding less background | In vitro ligation: more background colonies resulting from re-ligated vectors |
|
A-overhangs from PCR |
Not affected: 3' exonuclease eliminates A-overhangs created in the PCR step | Affected: 5' exonuclease enzymatic activity does not eliminate A-overhangs, reducing efficiency |
|
PCR primers |
15-nt overlap | Typically longer; 20–30 nt overlap |
Table 1 Comparison of In-Fusion Cloning with Gibson assembly-based cloning methods
Here, we demonstrate superior performance of the In-Fusion Snap Assembly Master Mix over the GeneArt Gibson Assembly HiFi Master Mix in two particularly difficult experiments: multi-fragment cloning and large fragment insertion. The side-by-side comparison shows In-Fusion Snap cloning overcame the most daunting challenges, producing high colony counts and consistently accurate constructs.
