title: Flexi vector cloning.
abstract: A protocol for ligation-dependent cloning using the Flexi Vector method in a 96-well format is described. The complete protocol includes PCR amplification of the desired gene to append Flexi Vector cloning sequences, restriction digestion of the PCR products, ligation of the digested PCR products into a similarly digested acceptor vector, transformation and growth of host cells, analysis of the transformed clones, and storage of a sequence-verified clone. The protocol also includes transfer of the sequence-verified clones into another Flexi Vector plasmid backbone. Smaller numbers of cloning reactions can be undertaken by appropriate scaling of the indicated reaction volumes.
authors.affiliation: Center for Eukaryotic Structural Genomics, Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA.
authors.forename: Paul G
authors.initials: PG
authors.lastname: Blommel
authors.forename: Peter A
authors.initials: PA
authors.lastname: Martin
authors.forename: Kory D
authors.initials: KD
authors.lastname: Seder
authors.forename: Russell L
authors.initials: RL
authors.lastname: Wrobel
authors.forename: Brian G
authors.initials: BG
authors.lastname: Fox
mesh_terms: D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000737:chemistry; D005822:Genetic Vectors; D005990:Glycerol / Q000737:chemistry; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D005990:Glycerol