Student Handout for Activity 3: Hands-on Laboratory Exercise
In this hands-on wet laboratory exercise, you will work together to:
· Grow a bacterial library
· Clone a gene of interest in bacteria
· Express a protein of interest in bacteria
· Purify a protein of interest from bacteria utilizing hydrophobic interaction chromatography
This lab uses a commercially available kit. See the Bio-Rad graphic below for a flow-chart of the lab exercises. This lab will demonstrate how you the scientist will clone a gene, grow it up in a bacterium, and then produce and purify the green glowing coqui silencing protein we are after!
Student Instructions:
Part 1: Streaking a bacterial plate to generate single colonies from a concentrated suspension of bacteria.
Materials:
Sterile inoculation loop
Poured agar plates n=2
Permanent marking pen
Bacterial library (shared amongst class)
37 °C Incubator (shared amongst class)
A small amount of the suspension will be used to create the library. Under proper conditions, a single bacterial cell can multiply and produce millions of identical daughter cells in 24 hours.
1. Turn your petri dishes upside down. Using a permanent marking pen, label your two agar plates with your lab group number or name.
2. Insert a sterile inoculation loop straight into the vial of the bacterial library without tilting the vial.
3. Remove the loop and streak for single colonies.
You will streak your plate four times, in four imaginary “quadrants” on each plate. Run the inoculation loop back and forth about 10 times in each of the quadrants. Turn your plate a quarter turn each time so that the entire plate covered as much as possible. See illustration:
4. When you are finished with a plate, cover it to avoid contamination.
5. Do the same thing with the second plate.
6. Place both plates UPSIDE DOWN in the incubator set at 37 °C overnight.
Part 2: Screening your colonies
Materials:
Streaked bacterial plates from last class n=2
Inoculation loops n=2
Culture tubes containing 2 ml growth media n=2
Marking pen
Test tube holder n=1
37 °C Incubator (shared amongst class)
UV light (may be shared amongst class)
1. Remove your streaked plates from the incubator and examine them in normal light.
Record your results in your laboratory notebook.
2. In a darkened area of the lab, observe your plates using a UV light. (Never look directly into the UV light and wear safety goggles to prevent damage to your eyes) Record your results in your laboratory notebook.
3. Identify the green colonies on one plate (you want colonies that are isolated and not touching other colonies), mark these colonies by turning the plate over and circling these colonies with a marker on the plastic.
4. Do the same on the other plate, but identify white colonies.
5. Get two 15 ml culture tubes that contain 2 ml of growth media. Label them both with your group or lab number then label one tube “+” and the other tube ”–.”
6. Using a sterile inoculation loop, lightly touch the end to the chosen green colony on first plate and gently scoop up the cells (careful not to get the agar!) Gently stir the inoculation loop in the 15 ml tube labeled “+”. Gently rolling the loop between your thumb and pointer finger will help get the whole colony in the culture tube.
7. Repeat this procedure, placing the white colony in the 15ml culture tube labeled “-.“
8. Cap your tubes and place them in the shaker. Let them incubate at 32 °C for 24 hours.
Part 3: Protein purification phase 1
Materials:
Microtube
Pipette (can be used repeatedly after rinsing)
Microtube rack
Marker
Beaker of water (for rinsing pipette)
TE buffer 1 vial (to be shared amongst class)
Lysozyme (rehydrated) 1 vial (to be shared amongst class)
Centrifuge
UV light
1. Label a microtube with your lab group initials or number
2. Remove the liquid cultures from the incubator and observe them in normal light and with the UV lamp (remember to protect your eyes!) Record what you see in your lab manual. Are there any color differences?
3. Using a clean pipette, transfer all of the liquid from the “’+” tube into the microtube and cap the tube.
4. Set aside the “-“ tube.
5. When your instructor calls you, spin the microtube in the centrifuge for 5 minutes at maximum speed. (Remember you have to balance the tubes!)
6. You will be able to visualize a pellet at the bottom of the tube as well as a liquid “supernatant” on top.
7. Open the tube and gently pour off the supernatant. The pellet will remain in the tube.
8. Observe the pellet using the UV lamp, (Remember to protect your eyes!) and record your observations in your laboratory notebook.
9. With a clean pipette, ad 250 µl of TE buffer to your tube. You want to resuspend the pellet into the new liquid. Do this by rapidly pipetting the liquid up and down several times.
10. With a cleaned pipette, add 1 drop of lysozyme to the resuspended pellet. Cap the tube and mix by flicking the tube with your finger. Lysozyme is an enzyme that will digest the cell wall of the bacteria.
11. Place the microtube in the freezer until the next class.
Part 3: Protein purification phase 2
Materials:
Microtube
Pipette (can be used repeatedly after rinsing)
Microtube rack
Marker
Beaker of water (for rinsing pipette)
HIC chromatography column n=1
Column end cap n=1
Waste beaker or tube
Binding buffer 1 vial (to be shared amongst class)
Equilibration buffer 1 vial (to be shared amongst class)
Centrifuge
UV light
1. Retrieve your microtube from the freezer and thaw is by rubbing it in your hands.
2. When the instructor calls, you will centrifuge your microtube (remember to balance!) for 10 minutes at maximum speed. During this time, label a new microtube with your initials or number.
3. While your sample is spinning, prepare the chromatography column by shaking it vigorously. To bring the hydrophobic beads to the bottom of the column, shake it one last time like a thermometer. Tap the column on the table to ensure the beads are settled at the bottom.
4. Remove the cap and snap off the bottom tab, allowing the liquid buffer to drain out of the column.
5. Using a pipette, Add 2 ml of Equilibration buffer to the top of the column 1 ml at a time, drain the buffer until it reaches the 1 ml mark on the column.
6. Replace the cap on the top and bottom and store at room temperature.
7. When your centrifuge has stopped, remove your tube and examine with a UV lamp (remember to protect your eyes!). You should be able to visualize a pellet at the bottom and a liquid supernatant on top. Note the colors of each in your lab notebook. This time, the pellet is the “trash” and we want to keep the supernatant. The pellet contains the cellular debris, while the supernatant will have our green glowing protein!
8. Using a clean pipette, transfer 250 µl of the supernatant into the new labeled microtube.
9. Using a cleaned pipette, transfer 250 µl of Binding buffer to the microtube. You should now have 500 µl of your supernatant plus binding buffer in the tube. Cap the tube and place it in the refrigerator.
Part 4: Protein purification phase 3
1. Get 3 collection tubes and label them with your initials. Label the tubes “1”, “2” and “3”. These tubes will be used to collect what runs through the chromatography column.
2. Retrieve your chromatography column and remove the cap from the top and bottom. Let the column drain completely into a waste container or over the sink.
3. When is has drained completely, gently place the column on collection tube #1. Do not force the column.
4. Using a pipette, gently load 250 µl of the supernatant plus binding buffer mixture into the top of the column by touching the pipette to the top of the inside of the column, letting the liquid drip down the side of the column wall. Let the entire volume flow into collection tube #1.
5. Visualize the column using UV light (remember to protect your eyes!) and note your observations in your notebook.
6. Move the column to collection tube #2. Using a cleaned pipette, add 250 µl of Wash buffer to the column utilizing the same technique as above. Allow the entire volume flow into the column.
7. Visualize the column using UV light (remember to protect your eyes!) and note your observations in your notebook.
8. Transfer the column to collection tube #3. Using a cleaned pipette, add 750 µl of TE Elution buffer to the column using the same technique as described above. Let the entire volume flow into the column.
9. Visualize the column using UV light (remember to protect your eyes!) and note your observations in your notebook.
10. Compare the collections tubes using UV light (remember to protect your eyes!) and note the differences in your lab notebook.
Student Handout for Activity 3Page 6 of 9 / Contextual Biology Integrated Projects
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