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TA0100

Sigma-Aldrich

TargeTron Gene Knockout System

Bacterial Gene Knockout

shipped in

wet ice

storage temp.

−20°C

General description

For detailed information, please visit the TargeTron home page: www.sigma-aldrich.com/targetron.

The TargeTron Gene Knockout System provides optimized reagents and protocols for the rapid and specific disruption of bacterial genes by insertion of group II introns. The method exploits the retrohoming ability of group II introns and utilizes a simple PCR step to re-target the TargeTron group II intron for specific insertion into the host genome. Utility of the technology has been demonstrated for prokaryotic genetic engineering, systems biology and functional genomics approaches.

Compared to existing knockout methodologies, the TargeTron system is quite rapid, simple and robust. The process involves loading the target gene sequence into the well-validated TargeTron algorithm on the Sigma website (www.sigma-aldrich.com/targetronaccess), using one or more of the output primer sets to mutate the TargeTron intron template via PCR, ligation of the fragment into the pACD4K-C linear vector, transformation of the host, induction of gene disruption, selection by kanamycin resistance (insertion of activated marker) and screening for the desired knockout.

Gene knockout using the TargeTron system has been validated in a broad range of bacterial strains such as Escherichia coli, Staphylococcus aureus, Clostridium perfringens, Shigella flexneri, Salmonella typhimurium, and Lactococcus lactis. The system may be modified for use in additional organisms.

Each TargeTron kit provides an access card and password for a specified number of designs (3 EA or 10 EA). The password gives the user access to the extensively validated TargeTron algorithm that predicts optimal intron insertion sites and designs primers for mutating the intron to insert into those sites. After selection of one or more desirable insertion points, the required oligos are ordered to begin laboratory procedures using the kit. Reagents in the 3 EA kit are sufficient for 3 designs, 12 reactions. Reagents in the 10 EA kit are sufficient for 10 designs, 40 reactions.

Application

TargeTron Gene Knockout System has been used to carry out site-directed mutation of genes within human monocyte-derived macrophages. It has also been used to disrupt the nuclease gene, nuc, in Staphylococcus aureus.

Features and Benefits

Targeted and permanent gene disruption
Simple, streamlined protocol; Knockouts in 3 days or less
Minimal screening to isolate mutants
>90% successful targeted insertion
No cell conjugation or specific host factor requirements

Components

System User Manual
TargeTron Access card (password for on-line target site selection)
Intron PCR Template
EBS Universal Primer
LacZ Control Primers
pACD4K-C Linear Vector (20 ng/μl)
JumpStart REDTaq® ReadyMix®
HindIII 20 U/μl
BsrGI 10 U/μl
10× Restriction Enzyme Buffer

Other Notes

To view the TargeTron Gene Knockout System animation, visit sigma.com/targetronanimation.

Legal Information

JumpStart is a trademark of Sigma-Aldrich Co. LLC
REDTaq is a registered trademark of Sigma-Aldrich Co. LLC
TargeTron is a trademark of InGex, LLC

Storage Class Code

10 - Combustible liquids

WGK

WGK 3

Flash Point(F)

Not applicable

Flash Point(C)

Not applicable

Certificate of Analysis

Enter Lot Number to search for Certificate of Analysis (COA).

Certificate of Origin

Enter Lot Number to search for Certificate of Origin (COO).

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