Mutagenesis using DpnI selection
- 최초 등록일
- 2014.09.13
- 최종 저작일
- 2014.08
- 6페이지/ 한컴오피스
- 가격 1,000원
목차
Ⅰ. Introduction
Ⅱ. Meterials
Ⅲ. Method
Ⅳ. Reference
본문내용
Ⅰ. Introduction
QuikChange® II Site-Directed Mutagenesis Kit allows site-specific mutation in virtually any double-stranded plasmid, thus eliminating the need for subcloning and for ssDNA rescue. In addition, It doesn't require specialized vectors, unique restriction sites, multiple transformations or in vitro methylation treatment steps.
The QuikChange® II site-directed mutagenesis kit is used to make point mutations, replace amino acids, and delete or insert single or multiple adjacent amino acids. The basic procedure utilizes a supercoiled double-stranded DNA (dsDNA) vector with an insert of interest and two synthetic oligonucleotide primers, both containing the desired mutation.
The oligonucleotide primers, each complementary to opposite strands of the vector, are extended during temperature cycling by DNA polymerase, without primer displacement. Extension of the oligonucleotide primers generates a mutated plasmid containing staggered nicks. Following temperature cycling, the product is treated with Dpn I. The Dpn I endonuclease (target sequence: 5'-Gm6ATC-3' is specific for methylated and hemimethylated DNA and is used to digest the parental DNA template and to select for mutation-containing synthesized DNA. (DNA isolated from almost all E. coli strains is dam methylated and therefore susceptible to Dpn I digestion.) The nicked vector DNA containing the desired mutations is then transformed into XL1-Blue supercompetent cells.
참고 자료
QuikChange® II Site-Directed Mutagenesis Kit
INSTRUCTION MANUAL (Catalog #200523, 500524) Stratagene