pREDET基因重组BAC克隆系统表达载体-BioVector NTCC质粒载体菌种细胞蛋白抗体基因保藏中心

pREDET基因重组BAC克隆系统表达载体

-BioVector NTCC质粒载体菌种细胞蛋白抗体基因保藏中心

BAC Subcloning Kit

Introduction

The completion of large DNA-sequencing projects, including the Human Genome

Project, has generated an extraordinary amount of primary sequence data. The next

major challenge is to investigate the components that make up a genome, and is

often called functional genomics. Escherichia coli vectors that can contain large

inserts, such as bacterial artificial chromosomes (BACs), P1 vectors and P1 artificial

chromosomes (PACs), offer several advantages for functional genomics. They can

carry sufficient DNA to encompass most eukaryotic genes, including all cis-acting

regulatory elements, as well as many eukaryotic gene clusters, prokaryotic regulons

and many complete viral genomes, in a single molecule. However, conventional

cloning methods rely on the use of restriction enzymes and in vitro purification steps,

which preclude engineering of large molecules. Consequently, the usefulness of such

molecules has been limited until recently.

Red®/ET® Recombination is the method that permits precise engineering of DNA

molecules of any size, including very large ones such as BACs or the E.coli

chromosome. It relies on homologous recombination in vivo in E. coli and allows a

wide range of modifications with DNA molecules at any chosen position.

Homologous recombination is the exchange of genetic information between two DNA

molecules in a precise and specific manner. These qualities are optimal for

engineering a DNA molecule regardless of its size. Homologous recombination

occurs through homology regions, which are stretches of DNA shared by the two

molecules that recombine. Because the sequences of the homology regions can be

chosen freely, any position on a target molecule can be specifically altered.

Red/ET Recombination utilizes homologous recombination and represents a

revolutionary DNA engineering platform that addresses the limitations found in

conventional methods.

BAC Subcloning kit

The BAC subcloning kit is designed to subclone DNA fragments of any size,

including very large fragments (> 20 kb) from any type of bacterial artificial

chromosomes (BACs, P1s, PACs) into a plasmid vector.

Contents of the kit:

1. pRedET (tcR): Red/ET expression plasmid (20 ng/μl, 20 μl)

2. minimal vector: PCR-template for generating a linear vector carrying a ColE1

origin plus ampicillin resistant (ampR) gene (50ng/μl, 20 μl)

3. minimal vector PCR-product: A ColE1 origin plus ampicillin resistant gene

(ampR) already flanked by homology arms to be used in the control reaction

for subcloning the mouse Hoxa11 gene (15kb) from a mouse BAC (100 ng/μl,

10 μl)

4. E. coli cells + control BAC + pRedET (tcR): Glycerol stock of E.coli strain

DH10B harboring the expression plasmid pRedET (tcR) as well as a

pBeloBAC11 derivate for the control experiment (500 μl, 25% glycerol)

5. pSub-Hoxa11: Glycerol stock of E.coli strain DH10B harboring the plasmid

which contains the mouse Hoxa11 gene (15kb) as positive control (500 μl,

25% glycerol)

Map of the Red/ET expression plasmid pRedET (tcR). Transformation of

E.coli hosts with this plasmid is selected for by acquisition of tetracycline resistance

at 30°C. Expression of the Red/ET Recombination proteins is induced by L-arabinose

activation of the BAD promoter at 37°C.

Flowchart of the experimental outline for subcloning a gene or part of a gene from a BAC into a plasmid.