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.
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. 10. Following the 15 minute incubation on ice, heat shock the cells using a water bath. 11. Use a sterile transfer pipet to add 250 uL Luria broth to each tube. Gently tap the
tubes to mix the LB with the cell suspension. Place the tubes in a test tube rack for a 5 to 15 minutes recovery. 12. Now you will remove some cells from each transformation tube and spread them on the plates. 13. Use a sterile transfer pipet to add 100uL of cells from the -plasmid transformation to the appropriate plate.
A. clam shell the lids and carefully pour 4-6 glass beads per plate
B. use a back and forth shaking motion to move the glass beads
C. let the plates rest for several minutes
D. remove the glass beads
15. Transfer 100uL of cell suspension from the +plasmid tube to each appropriate plate 16. Immediately suspend the cells
17. Wrap the plates together with tape and place the plates upside down in the incubator.
Purification of GFP by HIC Steps
1. Shake the culture tube to suspend the E.coli cells.
2. Use a micropipette to transfer 1mL of the overnight E.coli/GFP culture into a 1.5mL
3. Cap the tube and place it in a balanced configuration in the micro centrifuge rotor. Spin for one minute to pellet the cells. 4. Carefully pour off the supernatant. Do not disturb the green cell pellet. 5. Repeat steps 1-4 in the same 1.5 mL tube to pellet cells from a second 1 mL sample on
top of the first pellet. This will result in a large, green cell pellet. 6. Add 500mL of lyses buffer to the tube. Resuspend the cell. 7. Incubate the tube on ice for 15 minutes.
8. Microcentrifuge for 5 minutes.
9. Transfer 250mL of green cell extract into a clean 1.5mL tube. 10. Add 250mL of binding buffer to the tube containing 250mL of cell extract. Invert 11. Add 400mL of the cell extract/ binding buffer mixture to the tube of hydrophobic bead resin. Invert. 12. Microcentrifuge for 30 seconds.
13. Add 400mL of wash buffer to the...