A noncommercial polymerase chain reaction-based method to approach one hundred percent recombinant clone selection efficiency.
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.
Shareef
Mohammed M
MM
Weis Center for Research, Geisinger Clinic, Danville, PA 17822, USA. mmshareef@geisinger.edu
Dancea
Horatiu C
HC
Gross
Jessica L
JL
Myers
Tamara T
TT
Griggs
Wendy W
WW
Ahmed
Mansoor M
MM
Sheldon
David G
DG
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*
D004274:DNA, Recombinant