Introduction Chitobiase, from Vibrio harvey i, is a membrane bound lipoprotein involved in the degradation of chitin. Chitobiase is similar to and may share a common ancestry to the a-chain of human b-hexes-aminidase. Chitobiase is encoded by chb. In this experiment, a restriction map for restriction enzymes Eco R 1, Pst 1 and Hind III using Southern hybridization and restriction analysis of pRSG 192. pRSG 192 is a recombinant plasmid derived from the chb gene and pUC 19, a 2. 7 kb engineered plasmid which encodes for ampicillin resistance, a portion of the lac operon and a multiple cloning region.
The chb gene exists as a 3. 6 kb insert in the multiple cloning region of pUC 19. The major goals of Experiment One will be to isolate pRSG 192 from an overnight 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 Isolation Four microfuged tubes containing cell pellets representing 3. 0 ml of cells (2 x 1.
5 ml) from an overnight culture of E. coli were prepared. The supernatant fluid was discarded and each pellet was resuspended in 150 ul of TE buffer (10 mM Tris-HCl, pH 8. 0; 0.
1 EDTA). 300 ul of SDS (1% SDS, 0. 2 N NaOH) was added to each pellet. The tubes were placed on ice for five minutes, after which, 225 ul of ice-cold 3 M 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 recovered and transferred to new tubes. One volume of phenol / chloroform was added to each new tube. The tubes were shaken vigorously 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. The upper, aqueous phase of each tube was recovered and mixed with two volumes of 100% ethanol in a new tube. The tubes were microfuged for ten minutes and supernatants were discarded. The pellets were washed with 1 ml of 70% ethanol. The ethanol was poured off and pellets were air dried for 10 minutes.
The pellets were dissolved in 25 ul of TE, combined into one tube and treated with RNase.