Green Fluorescent Protein Lab
Title: Purification of Green Fluorescent Protein
Introduction: Transformation is used to introduce a gene coding for a foreign protein into bacteria. Hydrophobic Interaction Chromatography (HIC) is used to purify the foreign protein. Protein gel electrophoresis is used to check and analyze the pure protein. Research scientists use Green Fluorescent Protein (GFP) as a master or tag to learn about the biology of individual cells and multicultural organisms. This lab introduces a rapid method to purify recombinant GFP using HIC. Once the protein is purified, it may be analyzed using polysaccharide gel electrophoresis (PAGE).
Purpose: To illustrate the process of transformation and perform it using Green Fluorescent Protein.
Experimental Design: This experiment will achieve its purpose by allowing the student to perform a transformation following step by step instructions.
Procedure:
Laboratory Procedure for pGREEN Steps
1. Mark the sterile 15mL tubes with the correct labelings. (LB+plasmid, LB/Amp+plasmid).
2. Use a sterile transfer pipet to add 250mL of ice cold calcium chloride to each tube.
3. Place both tubes on ice.
4. Use a sterile plastic inoculating tube to transfer isolated colonies of E.coli from the starter plate to the +plasmid tube.
5. Immediately suspend the cells by repeatedly pipetting in and out with a sterile transfer pipet. Examine the tube against light to confirm that no visible clumps of cells remain in the tube or are lost in the bulb of the transfer pipet. The suspension should appear milky white.
6. Return the +plasmid tube to ice.
7. Use a sterile plastic inoculating tube to add one loopful of DNA to the +plasmid tube. immerse the loopful of plasmid DNA directly into the cell suspension and spin the loop to mix the DNA with the cells.
8. Return the +plasmid tube to ice and incubate for 15 minutes.
9. While the tubes are incubating, label your media plates.
Introduction: Transformation is used to introduce a gene coding for a foreign protein into bacteria. Hydrophobic Interaction Chromatography (HIC) is used to purify the foreign protein. Protein gel electrophoresis is used to check and analyze the pure protein. Research scientists use Green Fluorescent Protein (GFP) as a master or tag to learn about the biology of individual cells and multicultural organisms. This lab introduces a rapid method to purify recombinant GFP using HIC. Once the protein is purified, it may be analyzed using polysaccharide gel electrophoresis (PAGE).
Purpose: To illustrate the process of transformation and perform it using Green Fluorescent Protein.
Experimental Design: This experiment will achieve its purpose by allowing the student to perform a transformation following step by step instructions.
Procedure:
Laboratory Procedure for pGREEN Steps
1. Mark the sterile 15mL tubes with the correct labelings. (LB+plasmid, LB/Amp+plasmid).
2. Use a sterile transfer pipet to add 250mL of ice cold calcium chloride to each tube.
3. Place both tubes on ice.
4. Use a sterile plastic inoculating tube to transfer isolated colonies of E.coli from the starter plate to the +plasmid tube.
5. Immediately suspend the cells by repeatedly pipetting in and out with a sterile transfer pipet. Examine the tube against light to confirm that no visible clumps of cells remain in the tube or are lost in the bulb of the transfer pipet. The suspension should appear milky white.
6. Return the +plasmid tube to ice.
7. Use a sterile plastic inoculating tube to add one loopful of DNA to the +plasmid tube. immerse the loopful of plasmid DNA directly into the cell suspension and spin the loop to mix the DNA with the cells.
8. Return the +plasmid tube to ice and incubate for 15 minutes.
9. While the tubes are incubating, label your media plates.