Mutations Investigation: Bacterial Transformation Materials Mutated puc19 plasmid library XL-1 Blue competent E. coli cells 14 ml round-bottom tubes (1/transformation) Sterile cell spreaders (1/transformation) Pipettes and tips SOC medium (pre-warmed to 42 C) LB+ampicillin+IPTG+X-gal plates Sterile cell spreaders Shared Equipment Ice bucket Hot water bath (42 C) Incubating shaker (37 C, ~240 rpm) Warm incubator (37 C) Refrigerated incubator (5 C) Timer Sharpies Protocol Put on your safety glasses and gloves prior to beginning the protocol. 1. Label one 14 ml round-bottom tube clearly with your initials and today s date. Put it back into the ice bucket immediately it needs to remain cold for the next step. 2. Gently swirl the blue microcentrifuge tube containing XL-1 Blue competent E. coli cells, then carefully transfer 50 μl of cells from the blue microcentrifuge tube into your labeled 14 ml round-bottom tube using a P200 pipette and a fresh pipette tip. Eject the pipette tip into the biohazard waste container on your bench before passing the pipette on to the next student in your group. 3. Using either a P10 or P20 pipette with a fresh pipette tip, transfer 10 µl of the 20 pg/µl puc19 mutant plasmid DNA from the clear microcentrifuge tube to your labeled 14 ml round-bottom tube. Make sure the droplet of liquid goes into the cells in the bottom of your tube. Eject the pipette tip into the biohazard waste container on your bench before passing the pipette on to the next student in your group. 4. Gently swirl the tube to mix the contents, and immediately place back into the ice bucket. Leave the mixture of cells and plasmid on ice for at least 5 minutes. 5. The entire group of students sharing one ice bucket should complete this step together. Take the ice bucket and the timer with you to the hot water bath so that you can heat pulse your cells. Take your 14 ml round-bottom tube of cells and submerge it into the 42 C water bath up to the 1 ml mark for exactly 45 seconds. Be sure to use the timer to time this step the duration of the heat pulse is critical for obtaining the highest number of transformed cells. 6. Immediately place your 14 ml round-bottom tube back into the ice for 2 minutes.
7. Collect a microcentrifuge tube containing 1 ml of pre-warmed SOC medium from the hot water bath and return to your bench. SOC medium is a nutrient rich bacterial growth medium that is designed to help the bacteria recover from the transformation procedure. Using the P1000 pipette and a fresh tip, add 0.9ml of pre-warmed SOC medium to your tube of cells. Eject the pipette tip into the biohazard waste container on your bench before passing the pipette on to the next student in your group. 8. Immediately place your tubes into the incubating shaker, where the cells will be allowed to recover from the transformation process for 15 minutes. 9. After the 15-minute recovery period, collect a LB-agar plate with ampicillin, IPTG, and X- gal from the 37 C incubator. At your bench, label the plate with your name and the date. Use your P200 pipette with a fresh tip to measure 100 µl of the culture and spread it on the agar plate. Eject the pipette tip into the biohazard waste container on your bench before passing the pipette on to the next student in your group. Use a sterile cell spreader to spread the mixture uniformly over the surface of the plate. Be careful not to gouge the surface of the agar with the pipette tip or the spreader. 10. Place your labeled plate, lid down, into the 37 C incubator. The cells will be allowed to grow and form colonies overnight. 11. Be sure to clean up your bench!
Prelab Questions What pieces of Personal Protective Equipment (PPE) are required for all experiments? In several steps of the procedure, you will need to use different pipettes to transfer the correct volumes. For each of the following steps, indicate the volume you need to transfer and which pipette you will use. Step 3: Pipetting cells Step 4: Pipetting plasmid Step 8: Pipetting SOC media What does it mean for cells to become competent? Think about what your plate might look like if the plasmid did not have any mutations. Would you expect some, none, or all of the bacterial colonies to be blue? In this experiment, your cells will experience many different temperature changes. Write the temperature your cells will experience for each of the following steps. Incubating the cells before transformation (Step 5): C Heat pulsing your cells (Step 6): C Immediately after heat pulsing your cells (Step 7): C Recovery period in the incubating shaker (Step 9): C Incubation on plates at the end of the protocol (Step 11): C On the reverse side of this page, write a simplified version of this procedure in your own words. Feel free to use bullet points or diagrams.
Mutations Investigation: Monoculture Inoculation Materials LB-agar plate with transformed cells (from the previous laboratory session) 14 ml round-bottom polypropylene tubes LB medium with 100 µg/ml ampicillin (pre-aliquoted to 14 ml tubes) Sterile inoculation loop/needle tool Shared Equipment Incubating shaker (37 C, ~240 rpm) Sharpies Procedure Put on your safety glasses and gloves prior to beginning the protocol. 1. Label a new 14 ml round-bottom tube clearly with your initials and today s date. 2. Inspect the plate from your transformation and record the numbers of blue and white colonies in the Track Your Work section. 3. Select one colony that is well isolated from the others. Mark it by drawing a circle on the lid of the plate over the colony you have selected. Make sure you have properly aligned the lid before circling colonies. DO NOT write on the agar. 4. Record the color the colony you have selected in the Track Your Work section. 5. For the colony you selected, inoculate the 5 ml of media in one of the 14 ml roundbottom tubes as follows: a. Carefully open one sterile inoculating loop/needle. You will use the needle end of the tool. To prevent contamination, do not touch the tip of the needle or lay it down on the bench after you have removed it from the package. Needle Loop b. Gently touch the tip of the needle to the center of the colony you have selected. A typical colony is made up of billions of cells, so you will have no trouble collecting enough cells. There is no need to scrape up the entire colony. c. Dip the tip of the needle into the media in the tube labeled with the same number as the colony from which you have just collected cells. Swirl the needle around in the media to release cells into the solution. d. Place a cap on the tube containing media that you have just inoculated. The cap should not be completely closed, so that air can still circulate in and out of the tube. The used inoculating tool should be placed in the biohazard waste. 6. Put your inoculated cultures into the incubating shaker. They will be allowed to grow overnight.
7. Be sure to clean up your bench before you leave for the day! Track Your Work Transformation Plate Data Total number of colonies: Number of white colonies: Number of light blue colonies: Number of dark blue colonies: If you did not have any colonies, use this space to write your best guess as to why your transformation was unsuccessful:
Prelab Questions Why does the LB broth contain ampicillin? See the plasmid map shown below. Consider the effects of mutations in each of the labeled regions of the plasmid. In which region could a mutation result in a white colony? In what region could a mutation result in cell death? Briefly explain why. Why is it important to pick only one colony? What would happen to the culture if more than one colony was selected? If you pick a blue colony to inoculate a monoculture, do you expect to see a blue color in the monoculture cell suspension? Why or why not? On the reverse side of this page, write a simplified version of this procedure in your own words. Feel free to use bullet points or diagrams.
Mutations Investigation: Plasmid Isolation Materials Microcentrifuge tubes with: Solution 1 (blue label) resuspension buffer Solution 2 (red label) alkaline lysis buffer Solution 3 (purple label) neutralization buffer Solution 4 (yellow label) wash buffer Solution 5 (green label) elution buffer Spin columns 2 ml microcentrifuge tubes Pipettes and tips Liquid waste flask 0.5 ml Qubit tubes Qubit working solution Shared Equipment Vortex Centrifuge Sharpies Qubit fluorometer Procedure Put on your safety glasses and gloves prior to beginning the protocol. Part 1: Extract plasmid DNA 1. Retrieve your overnight 5 ml culture from the shaker. Label a microcentrifuge tube with your initials. 2. Using a P1000 pipette set to 900 µl, transfer a total of 1.8 ml of the culture into a clean, labeled microcentrifuge tube by pipetting 900 µl twice. Replace the tip every time you move to a new culture. The cells have a tendency to settle to the bottom of the tube, so be sure insert your pipette all the way to the bottom of the culture tubes to capture as many cells as possible. 3. Place your tubes in the centrifuge. Orient your tubes so that the tube hinge is facing straight out away from the center of the rotor. We will force the cells collect in the bottom of the tube by centrifugation at 16,000 g for one minute. 4. Collect your tubes from the centrifuge. You should notice a clump, or pellet, of cells at the bottom of your tube. 5. Decant the supernatant by inverting the tube and pouring into the liquid waste container. 6. As you did in step 3, transfer another 1.8 ml from each 5 ml overnight culture into the corresponding microcentrifuge tube. Be careful: do not mix cells from different cultures in the same tube. 7. Place your tubes in the centrifuge. Orient your tubes so that the tube hinge is facing straight out away from the center of the rotor. This will ensure the pellet forms in the same location as before. Everyone s tubes will be spun at 16,000 x g for one minute.
8. Collect your tubes from the centrifuge. Again, decant the supernatant by inverting the tube and pouring into the liquid waste container. You will need to do another spin to be sure to remove all traces of liquid from the sides of the tube. 9. Once more, place your tubes in the centrifuge. Make sure the tube hinge is facing away from the center. Everyone s tubes will be spun at 16,000 x g for one minute. 10. Collect your tubes from the centrifuge. Using a P20 pipette set to 20 ul, carefully remove all visible liquid from your pellet. Discard the supernatant into your waste container. It may be necessary to repeat this multiple times until all liquid has been removed. Make sure to use a fresh tip for each tube. 11. Collect your tubes from the centrifuge. Use the P200 pipette (with a fresh tip for each tube) to add 50 µl of Solution 1 to each cell pellet tube. Close the tubes. Solution 1 is a resuspension buffer that will help disperse the cells in preparation for lysing. If the cells remained in a tightly packed pellet, they would not all experience the same chemical conditions during the lysing step. Resuspension helps to ensure that the cells will all experience even exposure to the lysis solution in subsequent steps. 12. Resuspend the cells in Solution 1 by vortexing. Hold each tube on the vortexer for 10 seconds, take it off for 1 second, then hold it on the vortex again. Repeat this process for at least 1 minute, or until you no longer see any clumps of cells in the suspension. 13. Use a P200 pipette to add 100 µl of Solution 2 to the cell suspension. Close the tubes and mix by gently inverting about 8 times. Do not mix too vigorously, or the chromosomal DNA could break into smaller pieces that may co-purify with the plasmid DNA. Solution 2 will lyse the cells and cause them to break open. It is very alkaline (ph 12) and therefore causes proteins within the cell membrane to denature, and it also contains a detergent (SDS) that disrupts the structure of the cells. In addition, the high ph in this buffer will denature all the DNA (both chromosomal DNA and plasmid DNA) within the cells. 14. Neutralize the lysis mixture: Use a P1000 pipette to add 325 µl of Solution 3 to each tube. As before, close the tubes and mix by gently inverting the tubes about 8 times. Solution 3 is a neutralization buffer that restores the ph of the solution to about 7. The cells will remain disrupted. At neutral ph, the DNA is able to re-nature, but the chromosomal DNA is too big and messy to easily re-nature. The plasmid DNA is smaller, so it can easily renature into its double-stranded form. 15. Place your tubes in the centrifuge. Everyone s tubes will be spun at 16,000 g for one minute, at the same time. Meanwhile, label your spin columns with your initials and a number for each of your cultures. 16. Collect your tubes from the centrifuge. You may notice a compact, white pellet at the bottom of each tube. This pellet contains cell debris, including chromosomal DNA, and should be left behind in the tube. However, the relatively small plasmid DNA remains in the supernatant. Carefully pour the supernatant from each tube into the correspondingly labeled spin column. Discard the empty microcentrifuge tubes in the biohazard waste. Close the lids on the spin columns.
Part 2: Collect and quantify purified plasmid DNA 17. Place the spin columns in the centrifuge. Everyone s spin columns will be spun for 1 minute at 10,000 g, at the same time. The plasmid DNA now binds to the white silica membrane inside the spin filter. The neutralization buffer, which was applied in the previous step, contained a high concentration of salts. At high salt concentrations, DNA has a strong preference to bind to silica. However, other small components of the cell will pass through the silica membrane without binding. 18. Collect your spin columns from the centrifuge. For each spin column, temporarily remove the filter basket from the tube and pour the flow-through into the liquid waste container. Replace the filter basket into the tube. 19. Using a P1000 pipette, add 300 µl of Solution 4 to the spin column. Close the lids on the spin columns. Solution 4 contains ethanol, which helps to wash away impurities while maintaining the strong binding between the plasmid DNA and the silica membrane. 20. Place your tubes in the centrifuge. Everyone s tubes will be spun at 16,000 g for one minute, at the same time. Collect your tubes from the centrifuge. Carefully pour the flowthrough from each filter into the liquid waste. 21. Repeat step 20: Place your tubes in the centrifuge. Spin at 16,000 g for one minute. Collect your tubes. Pour the flow-through from each filter into the liquid waste. 22. Label three new microcentrifuge tubes with your initials and a number for each of your cultures. Transfer your filter baskets into the new tubes with the corresponding label. 23. Use a P200 pipette to carefully drop 50 µl of Solution 5 directly into the middle of the white spin filter membrane. Solution 5 is a low salt solution. At low salt concentrations, the binding between the plasmid DNA and the silica membrane is lessened, and the DNA will be released into the flow-through. 24. Place your tube in the centrifuge. Spin at 16,000 g for one minute. 25. Collect your tube from the centrifuge. The flow-through will contain purified plasmids. Dispose of the spin column in the biohazard waste container. 26. Label the top (not the side!) of a clear thin-walled tube for DNA quantification with your initials. 27. Add 198 µl of Qubit Working Solution to each tube. The Working Solution contains a fluorescent dye that binds to DNA and increases its fluorescence upon binding. 28. Add 2 µl of the purified plasmid sample to each tube, bringing the total volume to 200 µl. 29. Place your diluted DNA sample in the Qubit Fluorometer, take your measurement, and record the concentration on the class worksheet. The measured fluorescent signal should be proportionate to the amount of DNA in the sample.
Prelab Questions. Briefly define the role of each solution in the plasmid prep kit. Solution 1 Solution 2 Solution 3 Solution 4 Solution 5 Do we want to collect ALL the DNA from the cells? Why or why not? If two colonies are the same color, do we expect the same plasmid DNA sequence for both? Why or why not? On the next page there is a shorter version of the plasmid prep protocol. Using this protocol as a starting point, write additional notes to help you through the procedure. Or, use a separate sheet of paper to write your own version of the procedure entirely in your own words.
Abridged procedure Collect cells Add 1800 µl culture to tube Centrifuge, pour off supernatant Again, add 1800 µl culture to tube Again, centrifuge and pour off supernatant Centrifuge once more Remove all supernatant using a 20 µl pipette Resuspend cells Lyse cells Add 50 µl of Solution 1 Resuspend the cells by vortexing intermittently for at least 1 minute Add 100 µl Solution 2 and mix by gently inverting Remove cell debris Bind DNA Wash filter Elute Quantify Add 325 µl of Solution 3 and mix by gently inverting Centrifuge While avoiding the pellet, transfer the supernatant onto a spin column Centrifuge, discard supernatant Add 300 µl of Solution 4 Centrifuge, discard supernatant Centrifuge again, discard tube Transfer filter to fresh tube Add 50 µl of Solution 5 Centrifuge Discard the filter and store the flow-through containing your plasmid DNA. Add 198 ul of dye solution to a new quantification tube Add 2 ul of pure plasmid DNA to the solution and vortex Use the fluorometer to measure the concentration of DNA in your tube