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TA0100 Sigma

TargeTron Gene Knockout System

Bacterial Gene Knockout

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Properties

Related Categories Functional Genomics and RNAi, Molecular Biology, TargeTron Gene Knock Out System More...
shipped in   wet ice
storage temp.   −20°C

Description

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

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

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.

Other Notes

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

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.

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

Safety & Documentation

Safety Information

RIDADR 
NONH for all modes of transport

Frequently Asked Questions

Has the TargeTron system, Product TA0100,been validated in my organism?
The information on TargeTron validation in a specific organism can be found here.
How do I adapt TargeTron, Product TA0100, to my organism?
The information on adapting TargeTron to a specific organism can be found here.
Is it possible to knockout more than one gene with TargeTron, TA0100?Can I perform multiple rounds of gene knockouts?
Multiple knockouts can be generated without selection by using the TargeTron kit in conjunction with the pACD4 TargeTron Vector Set (no selectable marker) (TV0010). Multiple knockouts can be generated using removable selection by using the TargeTron Vector pACD4K-C-loxP (T2826) with the TargeTron kit.
How stable is the knockout using TargeTron, Product TA0100 after several generations?
Data in Lactococcus lactis mutants passaged for 80 generations illustrated that 100% of the mutants were stable.
How does the TargeTron, Product TA0100 work?Do you have any references?
The principles that TargeTron is based on can be found here.
Which document(s) contains shelf-life or expiration date information for a given product?
If available for a given product, the recommended re-test date or the expiration date can be found on the Certificate of Analysis.
How do I get lot-specific information or a Certificate of Analysis?
The lot specific COA document can be found by entering the lot number above under the "Documents" section.
How do I find price and availability?
There are several ways to find pricing and availability for our products.Once you log onto our website, you will find the price and availability displayed on the product detail page.You can contact any of our Customer Sales and Service offices to receive a quote. USA customers: 1-800-325-3010 orview local office numbers.
What is the Department of Transportation shipping information for this product?
Transportation information can be found in Section 14 of the product's (M)SDS.To access the shipping information for this material, use the link on the product detail page for the product.
How can I determine possible targets with the Targetron® algorithim? How do I get my primer designs?
Please click here to submit your genomic sequence of interest to Technical Service. We will let you know your available target regions according to the algorithim via email reply.If you are ready to obtain your primer designs, please provide your genomic sequence and your access code from your Targetron® kitin the email and we will provide your designs to you via email reply.
My question is not addressed here, how can I contact Technical Service for assistance?
Ask a Scientist here.
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Protocols & Articles

Related Content

TargeTron® Gene Knockout System Animation [VIDEO]

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. Unlike conventional DNA transpo...
Keywords: Genetic

pACD4 TargeTron® Non-Selection Vector Set

The pACD4 TargeTron Non-Selection Vector Set (TV0010) is a set of four linearized vectors intended for use with the TargeTron Gene Knockout System (TA0100) when the use of a selectable marker is not ...
Keywords: Polymerase chain reaction

Peer-Reviewed Papers
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References

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