A noncommercial polymerase chain reaction-based method to approach one hundred percent recombinant clone selection efficiency.
Anal Biochem
; 382(1): 75-6, 2008 Nov 01.
Article
en En
| MEDLINE
| ID: mdl-18674510
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ADN Recombinante
/
Reacción en Cadena de la Polimerasa
/
Clonación Molecular
Límite:
Humans
Idioma:
En
Revista:
Anal Biochem
Año:
2008
Tipo del documento:
Article
País de afiliación:
Estados Unidos