biochem lab 1 - Shoshana Levi Thursday AM Lab Report 1...

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Shoshana Levi Thursday AM Lab Report 1 Abstract The first lab utilized proper lab techniques with emphasis on sterilization. The growth of a bacterial population, namely E. coli , was monitored through a series of spectrophotometer readings. The growth curve obtained illustrated a typical log function character. The next lab was the initiation into the process of the overexpression of human carbonic anhydrase II (hCA2). The genomic DNAs of λ and ΦX174 phage were digested using Hind III and Hae III respectively, and analyzed using agarose gel electrophoresis. This provided standard markers later used in comparison to the desired hCA2 gene and pETBlue-2 vector. The hCA2 gene was amplified via PCR and analyzed through agarose gel electrophoresis. Comparison to the Hae III ΦX174 bands and construction of a standard size vs. migration distance curve allowed for the determination of the size and concentration of the PCR product. The PCR product was then purified in order to ensure the maximum effectiveness of its enzyme digestion. The PCR product (hCA2 insert) and pETBlue-2 vector were then digested and purified by running an agarose gel electrophoresis, excising the desired bands, one containing the insert and one containing the vector, and then extracting the DNA from the agarose slices. The purified insert and vector were ligated together in a theoretical 1:1 mole ratio for favored conditions. An agarose gel electrophoresis was run in order to confirm the actual concentrations of insert and vector used by comparison to the Hind III λ and Hae III ΦX174 (a standard curve of size vs. migration distance was constructed again). The
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ligation mixture was applied to a population of E. coli which were selected for transformation by growth on an agar plate containing the antibiotic ampicillin; only bacteria that obtained a plasmid will survive. Blue/white screening was used as a complementation test in order to differentiate between bacteria with the hCA2 gene (remain white due to an interrupted lacZ gene) and those without the hCA2 gene (turn blue in the presence of X-Gal). The white colonies can then be selected and used in future labs. Results A new culture of E. coli was initiated and incubated over the course of the lab period. At 20 minute intervals a one mL sample was taken and the absorbance was measured by a spectrophotometer at 600 nm. The resulting growth curve of absorbance vs. time (image 1) illustrated a lag phase in which cell growth was latent, and then a log phase where the population
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This note was uploaded on 04/16/2011 for the course BCH 4023 taught by Professor Allen during the Spring '10 term at University of Florida.

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biochem lab 1 - Shoshana Levi Thursday AM Lab Report 1...

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