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Design an experimental procedure that can be used to determine the localization of the protein encoded by this gene in the yeast cell. You know the DNA sequence of yfg and your starting material is purified yeast DNA. Select experimental steps and put them in the correct order.

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  • Localize yfg-GFP in (living) yeast with fluorescent microsope.Localize yfg-GFP in (living) yeast with fluorescent microsope:
    With fluorescent microscopy it is possible to visualize fluorescently labeled molecules inside tissues and cells.
  • Introduce expression vector with yfg-GFP into yeast.Introduce expression vector into yeast:
    A method for introducing foreign nucleic acid into yeast cells is electroporation. It uses a brief, high voltage charge which renders the cells permeable to the nucleic acid.
  • Analyse PCR product containing yfg.Analyse PCR product containing yfg:
    To check whether the PCR of yfg was successful, a sample of the PCR mixture is first analyzed on an agarose gel.
  • PCR of yfg.PCR of yfg:
    PCR stands for Polymerase chain reaction. Specific primers are used to amplify the DNA of yfg in a reaction tube, even in the presence of excess non-specific DNA.
  • Insert yfg into a yeast GFP expression vector.Insert yfg into a yeast GFP expression vector:
    Yeast expression Vector: A plasmid designed for production of a polypeptide in yeast. The protein is encoded on a piece of foreign DNA that has been inserted in the vector. The DNA cloning steps are always carried out in E coli and the vector provides a yeast specific promoter and in this case also the coding sequence for the green fluorescent protein (GFP). Often an inducer is used to initiate expression of the gene that is inserted into the vector.

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