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 restriction digests are not advisable.
*Dephosphorylation of the vector termini is not required; the vector will not recircularize in the In?Fusion Cloning reaction mix unless it carries 15-nt complementary overlaps at its termini.

2. Inverse PCR with primers positioned at the desired cloning site.
*Choice of cloning locus is flexible since suitable restriction sites are not required.
*Simultaneous PCR-mediated mutagenesis (deletion, insertion, base change) is possible.
*The 15-bp homologous overlaps can be added to the PCR-linearized vector.
*Preserve the integrity of the vector backbone by using a PCR 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 E. coli genomic DNA, or 15 kb of lambda DNA. It is compatible with two- or three-step PCR cycling, and exhibits minimal error rates on GC-rich templates.

Vectors linearized via restriction digest should be purified by a preparative agarose gel (covered with aluminum foil to prevent DNA damage). Electrophoresis should be done at a low voltage to ensure the separation of linear and circular (uncut) vector molecules.
Vectors linearized via inverse PCR should be treated with Cloning Enhancer (CE) to destroy the parental plasmid. CE-treated, PCR-linearized vectors may require additional purification by agarose gel electrophoresis if PCR byproducts are present in the linearized vector prep.

In-Fusion Cloning products