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581px-PBR322.svg

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This is a cloning plasmid that is used from E. coli. It was one of the first E. coli plasmids used. pBR322 is used to insert specific DNA into the organisms genome. The plasmid is 4361 base pairs long.

Cloning vector attributesEdit

The cloning vector requires a, origin of replication, selectable markers, a cloning site and/or a reporter gene. The cloning vector allows for a site for DNA to be inserted into the plasmid. The origin of replication allows the plasmid to replicate. pBR322 is a medium replication plasmid, which means it has 10-100 copies per cell. The selectable markers allow the cell that the plasmid is in, be selected for if it does or does not have the plasmid. The cloning site is a site that is where the new genes are inserted into the plasmid. The reporter gene is another way of testing if the cell picked up the modified plasmid or not. This is done by having all of the cells with the plasmid create a product to identify it from the other cells.

Selectable markerEdit

The plasmid pBR322 is very useful because of the selectable markers in it. It has two selectable markers, a resistance to Ampicillin and Tetracycline. This allows the cells with the plasmid to be selected for. Without the selectable marker, all cells would grow, not just the cells that contain the desired genes. Vector requires a selectable marker, which helps in identifying and eliminating non- transformants and selectively permitting the growth of the transformants. 

Cloning siteEdit

In the plasmid pBR322 there are over 40 major cloning sites. The cloning sited are where a restriction enzyme cuts the plasmid and inserts the new genes. This is what allows people to control what genes are inserted into the organism. Without the cloning site, you can't insert a desired gene.

Steps to use cloning vector pBR322Edit

The first thing that needs to be done is for the plasmid to be cut by the specific restriction enzyme a

Transformed

http://filebox.vt.edu/users/chagedor/biol_4684/Methods/transformed.gif

nd have the desired genes inserted. Note: where the break is in the plasmid so that the correct selectable marker is left in tact. Then the plasmid is added to a solution of cells which are then heat shocked to allow small pores to form letting the plasmids enter the cell. Then the cells are plated on a media with the proper selectable marker in it. The cells that grow into colonies have successfully picked up the plasmid.




ReferencesEdit

(1)"Cloning Vector." Wikipedia. Wikimedia Foundation, 31 Oct. 2013. Web. 12 Dec. 2013.

(2)"PBR322." Wikipedia. Wikimedia Foundation, 29 Nov. 2013. Web. 12 Dec. 2013.

(3)"Cloning and Molecular Analysis of Genes." Cloning and Molecular Analysis of Genes. N.p., n.d. Web. 12 Dec. 2013.

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