chm4302L-labreport1

chm4302L-labreport1 - L ab Report#1 Transformation of E...

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Lab Report #1 Transformation of E. Coli Nova Blue via recombinant plasmid of pET-Blue 2 vector and hCA2 gene insert Abstract : E. Coli Nova Blue was transformed with recombinant plasmid consisting of pET-Blue 2 vector and hCA2 gene insert. The recombinant plasmid was produced via a ligation reaction between the vector and insert. The E. Coli Nova Blue was then screened for recombinants via the blue/white screening method. Any cells that took up the recombinant plasmids grew to a white color, while cells that took up the plasmid lacking the insert grew to a blue color. Any cells that did not take up plasmid at all died. A total of 5 blue colonies grew and a no white colonies formed. This was most likely due to a failed ligation reaction of the hCA2 gene insert and the pET-Blue 2 vector. Results : Determination of growth curve for E. Coli cells. E. coli culture was grown in a flask with LB broth and spectrophotometric
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readings were taken at regular intervals. Absorbance was plotted against time in order to determine the shape of the growth curve and to determine where mid-log phase was for E. coli growth (as shown in figure 1 below). Since protein overexpression is best done at mid- log phase, we will know for future reference when to induce protein overexpression to get optimal results. Mid-log phase appeared to be at around 71 minutes from the initial spectrophotometric reading taken at 0 minutes. Amplification of hCA2 gene via Polymerase Chain Reaction and analysis of Size Standards. DNA digested via λ HindIII restriction enzyme and X174 DNA digested via Φ HaeIII restriction enzyme were analyzed via gel electrophoresis in order to be utilized as size standards for comparison of other DNA fragments later on(figure 2 ).
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Afterwards a PCR amplification of Human carbonic Anhydrase 2 gene (hCA2) was carried out in order to prepare enough hCA2 insert for future restriction enzyme digestion and ligation to pET-Blue 2 vector. Estimating concentration of PCR product and size of resulting fragments; PCR product purification and restriction enzyme digestion. The products of the PCR were
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analyzed via gel electrophoresis against a size standard ( HaeIII X174 DNA) in order to Φ determine the estimated size fragment of the PCR product (figure 3) . Only one band appeared in the lane for the PCR product (lane 3) and this band had a corresponding length of approximately
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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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chm4302L-labreport1 - L ab Report#1 Transformation of E...

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