MP-E2002 PLUS DRIVER

Background The ability to isolate and propagate large pieces of DNA has vastly expanded the study of gene networks and operons. Materials and Reagents 1. The Amp R carrying fragment from pLLX8 is amplified with primers that provide homology to the Target Sequences 1 and 2 spacer region sequences. T Spectinomycin dihydrochloride pentahydrate Sigma-Aldrich, catalog number: The ability to isolate and propagate large pieces of DNA has vastly expanded the study of gene networks and operons. 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.

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The Amp R carrying fragment from pLLX8 is amplified with primers that provide homology to the Target Sequences 1 and 2 spacer region sequences. A Ampicillin sodium salt Sigma-Aldrich, catalog number: Additionally, the spacer regions contain consecutive restriction digest sites that are used to puls the capture vector prior to recombination with target DNA sequences Figure 2.

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.

Background The ability to isolate and propagate large mp-f2002 of DNA has vastly expanded the study of gene networks and operons. A peer-reviewed protocol journal.

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The authors will be requested to answer your questions at their earliest convenience. The ability to isolate and propagate large pieces of DNA has vastly expanded the study of gene networks and operons. G Large glass beads, 5 mm Corning, catalog number: Your questions will be directed to the authors of the protocol. Original research article A brief version of this protocol appeared in: T Spectinomycin dihydrochloride pentahydrate Sigma-Aldrich, catalog number: ;lus use cookies on this site to enhance your user experience.

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MPMan Drivers Download

The flanking homology on these DNA sequences enables their assembly by homologous recombination when co-transformed into competent S. This can be accomplished via one round of PCR.

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 mmp-e2002 recombination: Once your questions are answered, you will be informed using the email address that you register with bio-protocol.

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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.

These restriction sites can be replaced with other restriction sites if desired by altering the primer sequence, i.

MPMAN MP-E2002 PLUS Manual

My bio page Edit user profile Signature Reset the password. No mp-f2002 fee; no access fee. 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. 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 E. News Become a Reviewer.

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.

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. Cammie F Lesser clesser mgh. By using our mp-e20002, you are agreeing to allow the storage of cookies on your computer.

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MPMAN MP-E PLUS MANUAL Pdf Download.

YS or other ura3 minus strain E. 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, catalog number: 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.

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.

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. This protocol was adapted from Reeves et al.

Bacterial pathogenicity islands and other contiguous operons can be difficult to clone using conventional methods due to their large size.