title: A noncommercial polymerase chain reaction-based method to approach one hundred percent recombinant clone selection efficiency.
abstract: Molecular cloning is an important procedure in molecular biology, but this is often a rate-limiting step and can be very time-consuming, possibly due to low ligation efficiency. Here, we describe a simple polymerase chain reaction (PCR)-based strategy to approach 100% selection efficiency. The post-ligation mixture containing the recombinant was subjected to insert-specific primer-mediated PCR amplification using a high-fidelity DNA Pfu polymerase generating a plasmid containing staggered nicks. The PCR mixture was then digested with endonuclease DpnI, which digests the methylated and hemimethylated parental DNA template. The nicked vector was transformed into XL1 blue supercompetent cells where the nicks were repaired, thus amplifying and selecting only the newly amplified recombinant clones.
authors.affiliation: Weis Center for Research, Geisinger Clinic, Danville, PA 17822, USA. mmshareef@geisinger.edu
authors.forename: Mohammed M
authors.initials: MM
authors.lastname: Shareef
authors.forename: Horatiu C
authors.initials: HC
authors.lastname: Dancea
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Gross
authors.forename: Tamara T
authors.initials: TT
authors.lastname: Myers
authors.forename: Wendy W
authors.initials: WW
authors.lastname: Griggs
authors.forename: Mansoor M
authors.initials: MM
authors.lastname: Ahmed
authors.forename: David G
authors.initials: DG
authors.lastname: Sheldon
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D003001:Cloning, Molecular / Q000379:methods*; D004274:DNA, Recombinant / Q000235:genetics* / Q000302:isolation & purification*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004274:DNA, Recombinant