Dolan DNA Learning Center

Materials For Pre-lab Activities Computers with internet access Student lab notebooks (printable worksheets) Sentence strips

________________________________________________________________________________________

Exploring Mutant Organisms Materials and Recipes

For Fly Observations 15 dissecting microscopes or magnifying glasses Clear plastic tape Student lab notebooks (printable observation sheets) 60 clear plastic Petri plates (100mm x 15mm) Wild-type fruit flies (Drosophila melanogaster): approximately 10-20 wild type flies per plate and edges sealed with clear tape Mutant fruit flies : Categorized into 3 mutation types including eye mutations (bar eyes, lobe eyes, white eyes, black eyes), wing mutations (scalloped wings, dumpy wings, curly wings, apterous wings, vestigial wings) and body mutations (sepia ebony, yellow body, antennapedia) in labeled (wing, body, eye) Petri plates with approximately 10-20 mutant flies per plate. For Roundworm Observations 15 dissecting microscopes or magnifying glasses 55 Clear plastic worm petri plates (60mm x 15mm) Student lab notebooks (printable observation sheets) NGM agar Wild-type roundworms (C. elegans): 10-20 wild type worms in each plate Mutant roundworms (C. elegans) : Categorized into 4 mutation types including blistered (Bli-1), dumpy (Dpy 1), roller (Rol-5), and uncoordinated (Unc-22) in labeled* Petri plates OP-50 E.coli (food for worms) *IMPORTANT: We suggest using a number key (#1 = Dumpy, #2 = Roller, #3 = Uncoordinated, #4= Blister) so that only the teacher knows the meaning of the labels. Purchasing Information Assorted mutated and wild type fruit flies - Carolina Biological Supply Company Petri dishes - VWR Scientific Dissecting microscopes - Leica E. coli - www.silencinggenomes.org (free) Wild type and mutant C. elegans - www.silencinggenomes.org (free) NGM agar for C. elegans - Carolina Biological Supply Company

__________________________________________________________________________________________
Developed at the Dolan DNA Learning Center. Copyright Cold Spring Harbor Laboratory.

Dolan DNA Learning Center

Recipes Bacterial Culture: Luria-Bertani (LB) Broth

________________________________________________________________________________________

Exploring Mutant Organisms Materials and Recipes

Makes one liter. Store at room temperature (indefinitely). 1. Weigh out: 10 g of tryptone 5 g of yeast extract 10 g of NaCl (m.w. = 58.44) Alternatively, use 25 g of premix containing all of these ingredients. 2. Add all ingredients to a clean 2-liter flask that has been rinsed with deionized or distilled water. 3. Add 1 liter of deionized or distilled water to flask. 4. Add 0.875 ml of 4 N NaOH. 5. Stir to dissolve the dry ingredients, preferably using a magnetic stir bar. 6. Dispense 100-mL aliquots into sterile 1500-mL bottles using one of the following methods A. Loosely put on the caps and Autoclave for 150 minutes at 121C. (To help guard against breakage, autoclave the bottles in a shallow pan with a small amount of water.) LB Agar Plates (makes 35 plates) Store at 4C (3 months) or room temperature (3 months). 1. Weigh out: 10 g of tryptone 5 g of yeast extract 10 g of NaCl (m.w. = 58.44) 15 g of agar Alternatively, use 40 g of premix containing all of these ingredients 2. Add all ingredients to a clean 2-liter flask that has been rinsed with deionized or distilled water. 3. Add 1 liter of deionized or distilled water. 4. Add 0.875 ml of 4 N NaOH. 5. Stir to dissolve dry ingredients, preferably using a magnetic stir bar. Any undissolved material will dissolve during autoclaving. 6. Cover flask mouth with aluminum foil, and autoclave solution for 15 minutes at 121C. 7. Allow the solution to cool just until the flask can be held by bare hands (55C). (If the solution cools too long and the agar begins to solidify, re-melt by briefly autoclaving for 5 minutes or less or heating in a microwave oven for a few minutes.) 8. While the agar is cooling, mark culture plate bottoms with the date and description of the media (e.g., LB). If using pre-sterilized polystyrene plates, carefully cut the end of the plastic sleeves, and save the sleeves for storing the poured plates. Spread the plates out on the lab bench. 9. When the agar flask is cool enough to hold, lift the lid of a culture plate only enough to pour the solution. Do not place the lid on the lab bench. Quickly pour in enough agar to just cover the bottom of the plate (~250 ml). Tilt the plate to spread the agar evenly, and immediately replace the lid. 10. Continue pouring agar into plates. Occasionally flame the mouth of the flask to maintain sterility. 11. To remove bubbles in the surface of the poured agar, touch the plate surface with the flame from a Bunsen burner while the agar is still liquid. 12. Allow agar to solidify undisturbed. 13. If possible, incubate the plate lid side down for several hours at 37C (overnight if convenient). This dries the agar, limiting condensation when plates are stored under refrigeration. It also allows the ready detection of any contaminated plates. 14. Stack plates upsidedown in their original sleeves for storage.

__________________________________________________________________________________________
Developed at the Dolan DNA Learning Center. Copyright Cold Spring Harbor Laboratory.

Dolan DNA Learning Center

________________________________________________________________________________________
C. elegans Culture NGM (Nematode Growth Media) Plates 1. Purchase NGM media Carolina Biological. 2. Pour the solution into 6 cm plastic petri dishes. Add enough to fill the plates about half full. If the plate has large bubbles (that are large enough for worms to crawl into), pop the bubbles by briefly flaming the surface with a Bunsen burner. Occasionally flame the mouth of the flask to maintain sterility. 3. Allow the plates to cool for at least one day before seeding them.

Exploring Mutant Organisms Materials and Recipes

Streak E. coli to obtain single colonies 1. Label the bottom of each LB agar plate with OP50 E.coli and the date. 2. Hold the inoculating loop like a pencil and sterilize the loop in the Bunsen burner flame until it glows red hot. 3. Remove the lid from the OP50 culture plate with your free hand. Do not place the lid on the lab bench. Hold the lid face down just above the culture plate to help prevent contaminants from falling on the plate or lid. 4. Stab the inoculating loop into a clear area of the agar several times to cool it. 5. Use the loop tip to scrape a visible cell mass from a bacterial colony. Do not gouge the agar. 6. Lift the lid of the new agar plate just enough to perform streaking as desribed below. Do not place the lid on the lab bench. a. Streak 1: Glide loop tip back and forth across the agar surface to make a streak across the top of the plate. Avoid gouging the agar. Replace the plate lid after each streak. b. Streak 2: Re-flame the inoculating loop and cool it by stabbing it into the agar away from the first (primary) streak. Draw the loop tip through the end of the primary streak and, without lifting the loop, make a zigzag streak across one quarter of the agar surface. c. Streak 3: Re-flame the loop and cool it in the agar. Draw the loop tip once through the end of the previous streak, and make another zigzag streak in the adjacent quarter. d. Streak 4: Re-flame the loop and cool it. Draw the tip once through the end of the previous streak, and make a final zigzag streak in the remaining quarter of the plate. e. Re-flame the loop, and allow it to cool before placing it on the lab bench. Make it a habit to always flame the loop one last time. Grow E. coli Overnight Cultures 1. Label a sterile 15-mL culture tube with OP50 and the date. 2. Use a 5-ml pipette to sterilly transfer 2 ml of LB broth into the tube. a. Ensure that the culture tube cap is unsnapped to the loose position. b. Attach a pipette aid or bulb to a pipette. Briefly flame the pipette cylinder. c. Remove the LB bottle cap using the little finger of your hand holding the pipette bulb. Flame the mouth of the LB bottle. d. Withdraw 2 ml of LB. Re-flame the mouth of the bottle, and replace the cap. e. Remove the cap of the labeled OP50 sterile 15-ml culture tube. Expel the sample into the tube, and replace the cap. 3. Use a sterile pipette tip to scrape a visible cell mass from a selected OP50 colony, and drop it tip-first into the culture tube. Replace the tube cap in the loose position. Alternatively, use an inoculating loop as follows: a. Sterilize the loop in the Bunsen flame until it glows red hot. Cool the loop by stabbing it several times into the agar near the edge of the plate. b. Use the loop to scrape a visible cell mass from a selected colony. Immerse the tip in the broth, and agitate it to dislodge the cell mass. c. Replace tube cap in the loose position. Re-flame the loop before setting it on the lab bench. 4. Incubate the tubes at 37C for 24 hours with continuous agitation or 48 hours without shaking.

__________________________________________________________________________________________
Developed at the Dolan DNA Learning Center. Copyright Cold Spring Harbor Laboratory.

Dolan DNA Learning Center

________________________________________________________________________________________

Exploring Mutant Organisms Materials and Recipes

Seeding Plates 1. Use a 5-ml pipette to sterilely seed each of the NGM-lite plates with OP50. a. Attach the pipette aid or bulb to the pipette. Briefly flame the pipette cylinder. b. Remove the cap from the OP50 overnight culture using the little finger of your hand holding the pipette bulb. Flame the mouth of the OP50 overnight culture. c. Withdraw 1 ml of overnight culture. d. Add 1 to 2 drops of culture to the center of the surface of each NGM-lite plates. The drops should occupy most of the plate surface, but should not touch the edges of the dish. 2. Grow the seeded plates face-up at room temperature for 24 hours. The bacterial lawn should be confluent and dry before any worms are added. Chunk C. elegans 1. Obtain a plate with worms. 2. Label the bottom of a fresh OP50-seeded NGM- liter plate with your group name or number, the date, and wild-type. 3. Examine both plates under the microscope for signs of bacterial or mold contamination - any growth of a different color or morphology (shape) from the OP50 lawn. Obtain a new plate if you detect any contamination. 4. Using your microscope, identify a region of the plate of wild-type worms that is densely populated with worms and eggs. 5. Sterilize a metal spatula or forceps. Dip the end of the implement into the ethanol beaker, and briefly pass it through a Bunsen flame to ignite the alcohol. Allow alcohol to burn off away from the Bunsen flame; the implement will become too hot if left in the flame. (Sterilization prevents cross- contamination with different C. elegans strains and non-OP50 bacteria.) CAUTION: Be extremely careful not to ignite the ethanol in the beaker. Do not panic if the ethanol is accidentally ignited. Cover the beaker with a Petri dish lid or other non-flammable cover to cut off oxygen and rapidly extinguish the fire. 6. Use the sterilized implement to cut a 1 cm x 1 cm (1/4 in x 1/4 in) piece of agar from the region identified in Step 4. 7. Carefully lift out the cut piece of agar with worms, and place it on the OP50 lawn of the fresh NGM-lite plate. Ideally, place the piece upside down to make it easier for the worms to crawl into the new bacterial food source. 8. Examine the new plate under the microscope to verify that you have successfully chunked the worms. Within a few minutes, worms should crawl from the agar chunk and be visible in the bacterial lawn. 9. Store the plate at 20C.For information on the care and cultivation of C. elegans, please reference: www.silencinggenomes.org.

__________________________________________________________________________________________
Developed at the Dolan DNA Learning Center. Copyright Cold Spring Harbor Laboratory.