What are vector linearization and purification options for In-Fusion?
What are vector linearization and purification options for In-Fusion?
Linearization options include:
1. Restriction digest with one or more restriction enzymes.
-For efficient In?Fusion Cloning, integrity of the linearized vector termini is essential. We recommend using high-quality restriction enzymes and performing digests over several hours. However, overnight restriction digest is 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.
-Vectors linearized via restriction digest should be purified by preparative agarose gel electrophoresis (covered with aluminum foil to prevent DNA damage). Electrophoresis should be performed at a low voltage to ensure the separation of linear and circular (uncut) vector molecules.
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 instead of the insert.
-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, and 15 kb of lambda DNA. It is compatible with two- or three-step PCR cycling, and exhibits minimal error rates on GC-rich templates.
3. 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.