Plasmid Extraction :: essays research papers

IntroductionChitobiase, from Vibrio harveyi, is a membrane strangle lipoprotein involved in the degradation of chitin. Chitobiase is similar to and may share a common ancestry to the a-chain of human b-hexos-aminidase. Chitobiase is encoded by chb. In this experiment, a parturiency map for restriction enzymes Eco R1, Pst1 and Hind III utilise Southern crosswalk and restriction synopsis of pRSG 192. pRSG 192 is a recombinant plasmid derived from the chb gene and pUC 19, a 2.7kb engineered plasmid which encodes for ampicillin resistance, a portion of the lac operon and a multiple copy section . The chb gene exists as a 3.6 kb insert in the mutiple cloning region of pUC 19.The major goals of Experiment One will be to sequestrate pRSG 192 from an all-night culture of E. coli, amplify a region of the chb gene using PCR, and to map restriction sites within the chb gene using restriction analysis and Southern hybridization. Methods Plasmid IsolationFour microfuge tubes containing c ell pellets representing 3.0ml of cells(2 x 1.5ml) from an overnight culture of E. coli were prepared. The supernatant fluid was discarded and each pellet was resuspended in 150ul of TE buffer(10mM Tris-HCl, pH 8.0 0.1 EDTA). 300ul of SDS(1% SDS, 0.2 N NaOH) was added to each pellet. The tubes were placed on glass for five minutes, after which, 225ul of ice-cold 3M potassium acetate(pH 4.8) was added. The tubes were again placed on ice for five minutes and subsequently microfuged for five minutes. The supernatants were aged and transferred to refreshful tubes. One volume of phenol/chloroform was added to each new tube. The tubes were shake smartly for two minutes and centrifuged for five minutes. The upper, aqueous phase was recovered and transferred to a new tube. One volume of chloroform was added to each tube. The tubes were vigorously mixed and microfuged for three minutes.