Does In-Fusion Cloning preserve the restriction site(s) used to linearize the vector?

In order to maintain the restriction sites, nucleotides can be added to the PCR primers between the template-specific portion and the 15-nt homologous overlap. The online Primer Design Tool allows you to choose whether or not to preserve the restriction sites. (The Primer Design Tool is compatible with Mozilla Firefox or Google Chrome web browsers, but not with Internet Explorer.)In-Fusion [...]

What cloning vectors are compatible with In-Fusion Cloning?

Any linear vector is compatible with In?Fusion Cloning. Linearization can be accomplished in one of the following ways: 1. Restriction digest with one or more restriction enzymes. *For efficient In?Fusion Cloning, the integrity of the linearized vector termini is essential. We recommend using high-quality restriction enzymes and performing digests over several hours. However, overnight [...]

What is the smallest DNA fragment compatible with In-Fusion Cloning?

The smallest insert successfully cloned with In-Fusion Cloning was a 50-bp synthetic oligonucleotide (including two 15-nt homologous overlaps with the vector termini). For In?Fusion Cloning of short synthetic oligos (between 50 and 150 bp), the suggested oligo-to-vector molar ratio is 5–15:1. Depending on oligo length, the optimal ratio must be determined empirically. Note: Non-phosphorylated [...]

What is the largest DNA fragment compatible with In-Fusion Cloning?

This technology has been optimized for cloning large fragments. DNA inserts up to 15 kb have been successfully cloned into pUC19 using In?Fusion Cloning (results confirmed by colony PCR screening).In-Fusion Cloning [...]

Do I have to purify the PCR-amplified insert and/or vector prior to performing the In-Fusion Cloning reaction?

Yes, the PCR-amplified DNA must be purified prior to In-Fusion Cloning. Following PCR, verify by agarose gel electrophoresis that your target fragment has been amplified. If a single band of the desired size is obtained, you can either spin-column purify (NucleoSpin Gel and PCR Clean?Up) or treat your PCR product with Cloning Enhancer (CE). However, if nonspecific background or multiple bands [...]