What's In-Fusion's compatibility with large vectors?

In-Fusion technology allows easy cloning of single or multiple DNA fragments directly into large vectors (e.g., adenoviral vectors at 32.6–36 kb) in a single reaction, without intermediate cloning into transfer/shuttle vectors.In-Fusion Cloning [...]

How to clone a gene of interest in-frame with a fluorescent protein (alternative protocol)

1. Choose any vector carrying a fluorescent protein. Linearize your vector either by restriction digest or inverse PCR. 2. Purify the linearized vector by gel extraction to ensure the isolation of only linear vector molecules. 3. Design In-Fusion PCR primers using the online Primer Design Tool. (The Primer Design Tool is compatible with Mozilla Firefox or Google Chrome web browsers, but not with [...]

What are the vector and insert properties related to transformation efficiency

Some vectors or inserts may contain reiterated sequences (e.g., retroviral or lentiviral LTRs, adenoviral ITRs, etc.). When working with such vectors, it may be necessary to optimize bacterial transformation to prevent rearrangements within the recombinant construct and increase its stability during rescue and amplification in E. coli. Follow heat shock with revival of the bacteria at 25–30°C [...]

What is the optimal transformation amounts for In-Fusion?

For transformation of chemically competent bacterial cells (e.g., Stellar Competent Cells), use 2.5 µl of undiluted In-Fusion Cloning reaction mix per 50 µl of cells. For transformation of electrocompetent cells, use 1 µl of 1:5 dilutedIn-Fusion Cloning reaction mix per 50 µl of cells. (Optional) For larger transformation volumes, 5.0 µl of undiluted, unpurified reaction mix can be transformed [...]

What are the bacterial strains not recommended for In-Fusion Cloning?

We do not recommend transforming In-Fusion reaction mixtures into any of the following: -E. coli strains lacking recA1 or endA mutations -E. coli strains engineered for a particular application (e.g., large-scale protein expression) -Gram-positive bacterial strains -Bacterial cells carrying nupG (deoR) mutations Note: If it is absolutely necessary to use a particular bacterial strain not [...]