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However, the traditionally used engineered plasmids for this purpose, such as bacterial artificial chromosomes BACs , while extremely useful, are limited by problems with DNA stability, copy number, and complex assembly requirements. As represented in Figure 1C, the I-SceI restriction sites present in the capture vector that flank the landing pad sequences can be used as described in Kuhlman and Cox to liberate and target the capture DNA to a previously engineered landing pad site. Targeting sequences 1 TS1 and 2 TS2: Original research article A brief version of this protocol appeared in: Once your questions are answered, you will be informed using the email address that you register with bio-protocol.

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Original research article A brief version of this protocol appeared in: The authors will be requested to answer your questions at their earliest convenience.

The methodologies described here were originally designed mp-f2002 capture and transfer the 31 kb of DNA operons that encode the Shigella flexneri type 3 secretion system onto the Escherichia coli chromosome Reeves et al. News Become a Reviewer. A Ampicillin sodium salt Sigma-Aldrich, catalog number: Vol 6, Iss 22, November 20, Olus Large glass beads, 5 mm Corning, catalog number: The Amp R carrying fragment from pLLX8 is amplified with primers that provide homology to the Target Sequences 1 and 2 spacer region sequences.

Additionally, the spacer regions contain consecutive restriction digest sites that are used to linearize the capture vector prior to recombination with target DNA sequences Figure 2. No publication fee; no access fee. This can mp-e202 accomplished via one round of PCR.

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A peer-reviewed protocol journal. Preparation of DNA to be recombined to generate the capture vector Overview of DNA fragments As outlined in Figure 1, the capture vector is assembled by combining the following 4 fragments of DNA via yeast endogenous homologous recombination: T Spectinomycin dihydrochloride pentahydrate Sigma-Aldrich, catalog number: Abstract Bacterial pathogenicity islands and other contiguous operons can be difficult to clone using conventional methods due to their large size.

Tetracycline resistant strain harboring an integrated landing pad cassette for use if transferring captured DNA into the chromosome Addgene, pluus number: However, if chromosomal integration of the captured region of DNA is desired, landing pad sequences that flank the targeting sequences should be incorporated into the PCR product. We use cookies on this site to enhance your user experience.

Targeting sequences 1 TS1 and 2 TS2: While we favor the use mpp-e2002 an engineered landing pls sequence, one could adapt the approach described below to target the insertion of the captured DNA to a specifically defined locus on the bacterial chromosome. My bio page Edit user profile Signature Reset the password.

Background The ability to isolate and propagate large pieces of DNA has vastly expanded the study of gene networks and operons. The objective of this section is to create a capture vector Figure 1a plasmid that can subsequently be used to capture large defined fragments of DNA Part II. These restriction sites can be replaced with other restriction sites if desired by altering the primer sequence, i.

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Your questions will be directed to the authors of the protocol. Bacterial pathogenicity islands and other contiguous operons can be difficult to clone using conventional methods due to their large size.

Integration of target DNA into a landing pad site on the bacterial chromosome Notes: YS or other ura3 minus strain E. This methodology has been successfully used to isolate and integrate at least 31 kb of contiguous DNA and can be readily adapted for the recombineering of Pluus.

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Materials and Reagents 1. Lpus using our website, you are agreeing to allow the storage of cookies on your computer. Generation of a capture vector Note: This protocol was adapted from Reeves et al. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.

However, the traditionally used engineered plasmids for this purpose, such as bacterial artificial chromosomes BACswhile extremely useful, are limited by problems with DNA stability, copy number, and complex assembly requirements.

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