What is the bacterial cell competency required for In-Fusion?

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

What are the PCR requirements for In-Fusion reaction?

PCR-amplified DNA must be purified prior to the In?Fusion Cloning reaction. This can be accomplished with one of the following options: 1. NucleoSpin Gel and PCR Clean-Up -Gel extraction enables selection of specific DNA fragments of the desired size from background PCR byproducts or other contaminants. -Column purification is appropriate if PCR did not produce a background smear. 2. Cloning [...]

What are the tips for amplification with In-Fusion PCR primers?

-The template-specific 3' end of the In-Fusion PCR primer should be 18–25 nt long, in order to ensure template amplification. -To determine the Tm of In-Fusion PCR primers, use independent software for PCR primer analysis, such as OligoAnalyzer 3.1 from IDT Technologies. Use standard Mg2+, Na+, and dNTP concentrations usually recommended for PCR. -For optimal amplification, perform the initial [...]

What are the compatible polymerases for In-Fusion?

In-Fusion Cloning is compatible with any PCR polymerase. 3' overhangs do not interfere with the cloning reaction. To ensure an error-free insert, use a polymerase with high proofreading activity, like PrimeStar Max DNA Polymerase (supplied with some In-Fusion Snap Assembly bundles). This polymerase is highly robust and accurate, enabling amplification of up to 6 kb of human genomic DNA, 10 kb of [...]

What’s the optimal length of homologous overlaps?

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