How should I plan my In-Fusion experiment

Successful In-Fusion Cloning reactions require 15-bp homologous overlaps at the termini of the cloning insert and linearized vector, or adjacent inserts if multiple inserts are to be joined simultaneously. We recommend increasing the overlap to 20 bp of homology for multiple-insert cloning. -These overlaps can be generated by PCR amplification or oligo synthesis of either of the cloning [...]

How can I ensure transformation efficiency and overall cloning efficiency?

In-Fusion Cloning is an all-in-one solution that maintains high transformation efficiency and also provides the highest possible level of cloning efficiency. While high transformation efficiency allows for a large number of transformed colonies, high cloning efficiency speaks to accuracy—ensuring that over 95% of transformants are correct, thus reducing the amount of time necessary to screen [...]

Can I use electroporation to transform the In-Fusion Cloning reaction mix?

1 µl of 1:5 diluted In-Fusion Cloning reaction mix can be electroporated into 50 µl of electrocompetent bacterial cells.In-Fusion Cloning products Care guide Caution [body] This care sheet provides general information only for handling Carolina™ bacterial cultures. When you work with bacteria, it is imperative that you use sterile techniques at all times. Failing to use sterile techniques can [...]

In an In-Fusion Cloning reaction, how many colonies should I expect from the negative control?

The negative control provided with the kit typically produces fewer than 5% blue colonies; the number of white colonies produced varies slightly depending on the strain. In general, fewer than 5% of the white colonies on an experimental plate contain background. It has been our observation that ?95% of the colonies on experimental plates are correct. This speaks to In-Fusion technology's high [...]

Can I transform In-Fusion Cloning reaction mixtures in amounts larger than what is recommended in the user manual?

We do not recommend this. Transforming the reaction mixture in an amount larger than what is stated in the user manual may be toxic to your cells. For transformation of the In-Fusion Cloning reaction, use 2.5 µl of undiluted, unpurified reaction mix per 50 µl of Stellar Competent Cells. (Optional) For larger transformation volumes, 5.0 µl of undiluted, unpurified reaction mix can be transformed [...]