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Unformatted text preview: Lab Report #2 Expression of hCA2 gene insert in recombinant pETBlue2 plasmid for synthesis of recombinant human carbonic anhydrase I I Abstract : Samples of bacterial cultures of transformed E. Coli Nova Blue cells (with pETBlue- 2/hCA2 recombinant plasmid and pETBlue-2 nonrecombinant plasmid) were resuspended, lysed, and microcentrifuged in order to isolate plasmid DNA from the rest of the cell. Afterwards the supernatant containing the plasmid DNA was purified and a restriction analysis of the isolated plasmids using PstI restriction enzyme was performed. Plasmids containing the hCA2 gene insert would have been cut at two separate PstI sites, resulting in two fragments (3948bp and 459bp). A plasmid that did not undergo a successful ligation with hCA2 insert would have been cut at the only available PstI site, resulting in only one fragment (3653bp). The resulting digests of the restriction analysis were analyzed via gel electrophoresis and the resulting bands were compared against cut Hind I I I DNA and HaeIII X174 DNA. The observation of 2 bands at around 3948bp and 459bp respectively, would indicate the presence of plasmid with the hCA2 gene, whereas the observation of one band at around 3653bp would be indicative of plasmid containing no hCA2 insert. In one lane, the sample produced two bands, one at 3750bp and the other at 470bp, indicating the sample placed in that lane had the recombinant plasmid. In the other lane, only one band appeared and it was around 3400bp, indicating that the sample had only the original plasmid in it. Next, sample of plasmid containing the hCA2 gene insert was combined thoroughly using pipet t ip with E. Coli Tu ner (DE3) pLacI cells that were on ice and afterwards heat shocked. SOC growth medium was then added to the mixture. The cells were incubated for an hour and then screened for recombinants via the white/blue screening method. Any colonies that took in the recombinant plasm id grew white and any colonies that took in the non-recombinant plasm id grew blue. Cells that did not take up either did not grow. A total of 8 white colonies and 0 blue colonies grew, which was not surprising given that only recombinant plasmid was used in the transformation reaction. Afterwards, a sample of a recombinant colony was inoculated with LB + ampicillin, , and glucose until the culture reached an O.D. 600 value between 0.6-1.0. Afterwards, the cells were pelleted, supernatant removed, then a sample was taken of the cells, then IPTG was added to the remainder of the cells in order to induce protein expression of the hCA2 gene insert. Samples were taken at one hour intervals for a total of 3 hours in order to observe protein expression progression with time. Each time- point sample was centrifuged and washed with EDTA. Afterwards an SDS-PAGE gel was prepared to analyze each time-point sample in order to determine at what time was it optimal to harvest cells in order to maximize the amount of human carbonic anhydrase I I...
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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.
- Spring '10