Cloning of Enhancer of Zeste Homolog 2 in Forward Orientation Into Escherichia Coli Using Histidine-Tagged Pbluescript Ii Ks+.

4357 Words Mar 30th, 2013 18 Pages
Title: Cloning of Enhancer of Zeste Homolog 2 in forward orientation into Escherichia Coli using histidine-tagged pbluescript II KS+.
Enhancer of Zeste Homolog 2 locus is intensely over expressed in breast and prostate cancer and it’s been established that its promoter inhibition by p53 has led to reduced cell proliferation and invasion (Bracken, 2003; Xiao, 2011). Objective is to clone a forward orientated EZH2 insert into a his-tagged pbluescript. Cloning EZH2 into a histidine-tagged pbluescript in a forward orientation potentially allows isolation of protein via Affinity Chromatography or Chromatin Immunoprecipitation therefore its role, effects and targets in the genome can be established. Resultant Recombinant plasmids in
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DNA being a negatively charged molecule will move to the positive anode and its size determines its pace (Brown, 2010). Before using T4 ligase in ligations the vector was treated with alkaline phosphotase, diminishing the chances of parental self ligation. T4 ligase from T4 bacteriophage (with ATP and Mg2+) catalyses the formation of the phosphodiester bond by a nucleophillic attack of the adenyl residue attached to the 5 prime phosphate group of the insert by the 3 prime hydroxyl group of the vector (Sambrook and Russell, 2001).
The recombinant plasmid is then placed into competent Escherichia Coli, These cells are then spread on an agar plate, within the host cell the recombinant plasmid multiplies and then the host cells divides via Binary fission and copies of the recombinant plasmids are passed to its progeny and additional vector replications occurs. Large numbers of cell division leads to the formation of colonies, each cell in the colony contain one or two copies of the recombinant plasmid, hence the gene is now said to be cloned (Brown, 2010).
The blue/white colour of recombinant clones depends on if LacZ´ selectable marker was inactivated by the insert (EZH2), encoding a non functional segment of β-galactosidase which when functional degrades Xgal to 5-bromo-4-chloro-indoxyl producing the blue colour (Brown, 2010).
The experimental goal using these various techniques was to successfully insert the gene (EZH2) into

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