MP-E2002 PLUS DRIVER DOWNLOAD

The inclusion of flanking landing pad sequences does not preclude the propagation of the DNA of interest on an autonomously replicating plasmid, but rather affords the opportunity to subsequently introduce the captured DNA onto a defined site on the bacterial chromosome. Once your questions are answered, you will be informed using the email address that you register with bio-protocol. While we favor the use of an engineered landing pad 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. By using our website, you are agreeing to allow the storage of cookies on your computer. YS or other ura3 minus strain E. Targeting sequences 1 TS1 and 2 TS2: We use cookies on this site to enhance your user experience.

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Generation of a capture vector Note: YS or other ura3 minus strain E. Background The ability to isolate and propagate large pieces of DNA has vastly expanded the study of gene networks and operons.

MPMAN MP-E2002 PLUS Manual

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.

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T Spectinomycin dihydrochloride pentahydrate Sigma-Aldrich, catalog number: Tetracycline resistant strain harboring an integrated landing pad cassette for use if transferring captured DNA into the chromosome Addgene, catalog number: Additionally, the spacer regions contain consecutive restriction digest sites that are used to linearize the capture mp-r2002 prior to recombination with target DNA sequences Figure 2.

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These restriction sites can be replaced with other restriction sites if desired by altering the primer sequence, i. While plud favor the use of an engineered landing pad 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. The flanking homology on these DNA sequences enables their assembly by homologous recombination when co-transformed into competent S.

The Mmp-e2002 R carrying fragment from pLLX8 is amplified with primers that provide homology to the Target Sequences 1 and 2 spacer region sequences. The ability to isolate and propagate large pieces of DNA has vastly expanded the study of gene networks and operons.

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Alternatively, incorporating constructs directly into the bacterial chromosome provides advantages by both reducing variations in gene expression arising from the presence of multiple gene copies and ensuring stable maintenance of genes, while also avoiding the need for antibiotic selection. A Ampicillin sodium salt Sigma-Aldrich, catalog number: Vol 6, Iss 22, November 20, 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: Cammie F Lesser clesser mgh.

You are highly recommended to post your data including images for the troubleshooting. 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.

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Original research article Mp-e2002 brief version of this protocol appeared in: Bacterial pathogenicity islands and other contiguous operons can be difficult to clone using conventional methods due to their large size. Materials and Reagents 1. No publication fee; no access fee. The authors will be requested to answer your questions at their earliest convenience.

This can be accomplished via one round of PCR. This methodology has been successfully used to isolate mp-e20022 integrate at least 31 kb of contiguous DNA and can be readily adapted for the recombineering of E.

Abstract Bacterial pathogenicity islands and other contiguous operons can be difficult to clone using conventional methods due to their large size. Your questions will be directed to the authors of the protocol. This protocol was adapted from Mp-ee2002 et al.

Targeting sequences 1 TS1 and 2 TS2: The methodologies described here were originally designed to 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. The inclusion of flanking landing pad sequences does not preclude the propagation of the DNA of interest on an autonomously replicating plasmid, but rather affords the opportunity to subsequently introduce the captured DNA onto a defined site on the bacterial chromosome.