What bacterial strains are compatible with In-Fusion Cloning?

In-Fusion Cloning requires bacterial cells with competency no less than 108 cfu/µg supercoiled DNA. -Stellar Competent Cells (included in all current In-Fusion Cloning kits), as well as any general purpose cloning E. coli strain, should be compatible with In-Fusion Cloning. *Stellar Competent Cells have been validated for cloning and amplification of large vectors (e.g., BACs, fosmids) and [...]

Can I use TOP10 cells for In-Fusion Cloning?

TOP10 cells or their derivatives (e.g., ccdB Survival 2T1R E. coli), and related strains (e.g., DH10B, MC1061) are suboptimal for In?Fusion Cloning, resulting in a lower number of recombinant clones. This may be of particular concern if you are performing multiple-fragment cloning, or using a low-copy number vector. We recommend using Stellar Competent Cells, which are optimized for use with In [...]

Will cloning efficiency increase if I use a longer incubation time for the In-Fusion Cloning reaction?

No, an increase in the In?Fusion reaction time is not recommended. It may generate uneven single-stranded regions at the ends of the cloning insert and vector, resulting in inefficient annealing of the homologous overlaps, thus reducing cloning efficiency.In-Fusion Cloning [...]

Can I modify the length of the homologous overlap? Will a longer overlap improve In-Fusion Cloning efficiency?

Current In-Fusion Cloning reaction conditions favor a 15-bp homologous overlap for single-insert cloning, and a 20-bp homologous overlap for multiple-insert cloning. We do not recommend using overlaps shorter than 12 bp or longer than 21 bp.In-Fusion Cloning [...]

What are the recommended insert-to-vector molar ratios for In-Fusion Cloning?

In-FusionCloning uses a very robust enzyme, and allows highly efficient cloning in most situations. General recommendations on insert/vector quantities are included in all current In-Fusion Cloning user manuals. -To ensure optimal results under standard conditions, or when performing single- or multiple-fragment cloning, use an insert-to-vector ratio of 2:1. *The molar ratio of each of the [...]