Materials

• Large plastic or glass tub
• NaOH pH drops
• Alt. Sodium Bicarbonate
• Hitop H-608 Self-Regulating Custom Heater (opt.)
• MgSO₄ · 7H₂O
• Alt. CaO
• K₂CO₃
• Alt. CaCO₃
• African Cichlid Substrate (opt.)
• Alt. Sedimentation
• Deionized Water

Procedure

1. Fill the large tub with deionized water around half way. Record accurately the volume of water added.
2. Measure initial pH using a pH meter. Repeat NaOH drops until the pH is exactly between 7.7 pH and 8.6 pH.
3. Weigh out 172.4 mg/L MgSO₄ · 7H₂O multiplied by the liters of water plus one half and dissolve it in a 500 ml solution of deionized water.
4. Alternatively, use 70 mg/L of CaO.
5. Weigh out 271.1 mg/A K₂CO₃ multiplied by the liters of water plus one half and dissolve it in the same 500 mL solution.
6. Alternatively, use 196.328 mg/L of CaCO₃.
7. Add the 500 ml solution to the large tub.
8. Place the electronic heater in the water and set it to between 24°C and 29°C.
9. Alternatively, use no heater if room temperature is accurate.
10. Add a bed of commercial African Cichlid Substrate at 1.5 lb for each gallon of water in the microcosm.
11. Alternatively, use any other non-enriched sedimentation.
12. Situate the microcosm near a window with lots of sunlight, or outside under a transparent enclosure to protect from outside contaminants.

Materials

• Lake Malawi microcosm
• W303 yeast
• Lettuce
• Multiple Biomphalaria glabrata snails
• Alt. Pomacea bridgesii snails
• Warm water

Procedure

1. Refrain from feeding the snails for 6 hours prior to experimentation.
2. Prepare a W303 yeast plate, and multiple CM Broth agar plates.
3. Situate the microcosm with a large portion of sedimentation above the water level.
4. Place the snail and lettuce in the microcosm to form an equilateral triangle between two pieces of lettuce situated on the shore of the sedimentation, and one B. glabrata snail in the water. Each object should be 18 cm away from each other.
5. Using a toothpick, pick at least 15 W303 colonies and set them on top of one of the lettuce pieces (randomly selected).
6. Give at least 3 minutes for the snail to move towards a lettuce piece.
7. If snails are asleep, they can be stimulated via warm water.
8. After the snail has made contact with a lettuce piece, record whether they selected the yeast or non-yeast option. Reset the microcosm using new snails and lettuce pieces.
9. Repeat the procedure for at least 20 total trials.
10. For control testing, repeat the procedure but only assign one of the lettuce pieces to be the yeast option without actually placing yeast on it. Record the number of times yeast is selected to determine the proportion of yeast preference possible by random chance.

Materials

• Lake Malawi microcosm
• W303 yeast

Procedure

1. As a control, sample the microcosm water preemptively and plate on a CM Broth agar plate.
2. Insert 5 ml of yeast media in YPD Broth or at least 15 W303 colonies into the microcosm water.
3. Immediately sample and plate the water after insertion of yeast.
4. Wait 24 hours and then plate another sample of microcosm water. Repeat for however many and however long time intervals desired.
5. Record the growth on each plate in a qualitative positive or negative analysis on if the yeast is still surviving in the microcosm after any given time.

Materials

• Compounds: continentalic acid, sanguinarine chloride, plumbagin (from ThermoFisher)
• Dimethyl sulfoxide (DMSO)
• Inverted microscope (Zeiss Axiovert A1)
• Polypropylene, round-bottomed 96-well plate
• OxyFree oxygen absorber sachets
• Live schistosome miracidia
• DMEM/F-12 medium
• Opti-MEM medium

Procedure

1. Dissolve compounds as 1 mM stocks in dimethyl sulfoxide (DMSO) and store at -20°C until use.
2. Place miracidia in a 2:1 mixture of DMEM/F-12 and Opti-MEM medium.
3. Leave miracidia at 26°C for 18 hours in a small plastic container containing three OxyFree oxygen absorber sachets.
4. Collect newly transformed sporocysts from the containers into a 1.5 ml tube.
5. Prepare serial dilutions of the compounds in DMSO at 200X of their final assay concentrations, over seven concentrations ranging from 5 to 0.01 µM.
6. To each well of a 96-well assay plate, add 1 µl of 200X concentrated compound followed by 190 µl of fresh transformation medium.
7. While applying constant mixing, add 10 µl of suspended sporocysts to give a total volume of 200 µl to each well.
8. Place the plate in the same plastic container containing three OxyFree oxygen absorber sachets.
9. Store the plastic container at 26°C for 24 hours.
10. Then examine sporocysts using an inverted microscope for the presence of an intact tegument (outer surface) versus those with disrupted tegument, indicating the survival of sporocysts. Note that Sporocysts are poorly motile so motility is not used as a primary indicator of vitality.

Materials

• Wide Orifice Tips
• "Hockey Stick" Inoculation Loops
• SOC
• High-Efficiency NEB5 Alpha Cells (stored at -80°C)

Procedure

1. Put ice in an ice bucket.
2. Transfer a tube of E. coli competent cells (high-efficiency NEB5 alpha cells) from -80°C to ice.
3. Label a PCR strip and place it on ice for pre-cooling the PCR strip. Close the lid of the parts to be used.
4. Place the DNA sample used for transformation on ice.
5. Keep the tube of competent cells, the empty PCR strip, and the DNA sample on ice for 5 minutes.
6. Using a wide-orifice tip (to be gentle to the cells) and a pipette set to 10 µl, pipette the cells up and down 5 times to make the suspension even.
7. Add 2 µl of the E. coli cell suspension to a tube within the PCR strip.
8. Using an inoculation needle, pin-transfer the DNA sample into the cell suspension, Tap the mixture inside the tube using the inoculation needle 10 times to mix.
9. Keep the PCR strip on ice for 30 minutes.
10. Return the tube of competent cells to -80°C.
11. Use a thermocycler and set the temperature of the heat block to 42°C.

Materials

• Dpn1 Enzyme (from -20°C: keep on ice)
• PCR Product
• 37°C Incubator

Procedure

1. Add 2 µL of the enzyme to the completed PCR reaction. (1 µL if 20µL, 2 µL if 50 µL).
2. Use P20 set to -5µL, and pipette up and down 7 times. (P20 to 10µL if the reaction is 20µL, P200 to 25µL if the reaction is 50µL).
3. Incubate at 37°C for 1-3 hours. prior to removing from the fabric

Materials

• 1L Distilled Water
• 20 g Powdered Agar
• 28.4g CM Broth minus URA
• 2 L Conical Flask
• Petri Dishes

Procedure

1. Pour 1 L of distilled water into the conical flask. Measure 20g and 28.4g of Powdered Agar and CM Broth in weighboats.
2. Pour the CM Broth powder into the conical flask. Dissolve the powder by shaking the flask.
3. Shake the flask while pouring in the Agar powder. Mix the flask with a large magnet spinner for 3 minutes at 1000rpm, or until all agar clumps are dissolved.
4. Autoclave on an autoclavable tray at 121°C, S:20. Leave in autoclave for the full cycle (52 minutes).
5. Take the media out to cool down for 20-30 minutes after the autoclave is done.
6. While cooling the media, set the petri dishes under the fume hood and sterilize with UV light for 15 minutes. Pipette 25 mL of media into each dish, avoiding any bubbles.
7. Leave the plate lids off until their moisture dissipates, and wait for agar to dry. Store at 4C.

Materials

• Yeast Lysis Buffer
• 20 mM HEPES, pH 7.6
• 50 mM NaCl
• Yeast Suspension Buffer
• Zymolyase 20T
• Beta-mercaptoethanol

Procedure

1. Spheroplast Formation
2. Pellet yeast cells (bacterial culture, OD600 1.5-2.0) by centrifugation at 200-500 x g for 5-10 minutes. Suspend the cell pellet in an equal volume of Yeast Suspension Buffer. Add 1 µl of ß-mercaptoethanol per 100 µl yeast suspension.
3. Gently pipette up and down until the cell suspension is homogeneous. Incubate the suspension for 5 minutes at 4°C. Gently pipet again to suspend the cells.
4. Flick the vial containing Zymolyase® to mix the solution. Add 10 µl Zymolyase® for each 100 µl cell suspension in Yeast Lysis Buffer. Gently mix the content.
5. Incubate the suspension at 37°C for 30-60 minutes. Note: (OPTIONAL). Lysis can be monitored by taking 25 µl of suspension and mixing with 1 ml Yeast Lysis Buffer and reading the optical density at OD 800nm.
6. At the end of incubation, centrifuge the suspension at 10,000 x g for 5 minutes. Remove and discard the supernatant carefully, leaving the spheroplast pellet in the tube.
7. Spheroplast Lysis
8. For lysis, suspend the spheroplast pellet in an appropriate volume of the Yeast Lysis Buffer (2-3 times the volume of spheroplast pellet). Pipet the suspension up and down a few times. Vortex periodically and incubate on ice for 30 minutes. The lysis may be further facilitated by incubating the cells for 1-3 minutes at 37°C or a brief sonication step. Sonication is necessary for shearing genomic DNA. Please note, the higher Yeast Lysis Buffer to spheroplast pellet ratio the better the cell lysis.
9. Centrifuge the lysed cells at 20,000 x g, for 30 minutes at 4°C and collect the clear lysate. Note: Additional volume of Yeast Lysis Buffer can be purchased separately for downstream applications, e.g. chromatography and dialysis, etc.

Materials

• 0.2 M Lithium acetate 1% SDS solution
• Ethanol 96-100^ and 70%
• Heat Block

Procedure

1. Place one yeast colony from the plate or spin down 100-200 µL of liquid yeast culture (Od600 ~0.4). Suspend cells in 100 µL of 200 mM LiOAc, 1% SDS solution.
2. Incubate for 5 minutes at 70 C.
3. Spin down DNA and cell debris at 15000 g for 3 minutes
4. Wash the pellet with 70% ethanol.
5. Dissolve the pellet in 100 µL of H2O or TE and spin down cell debris for 15 seconds at 15000 g.
6. Use 1 µL of supernatant for PCR.

Materials

• 0.8 g Agarose (for 0.8% agarose)
• TAE 0.5x Buffer
• 6 µL DNA Ladder (mixed w/ Loading Dye)
• 1 µL DNA samples
• Gel Casting Tray
• Gel Chamber
• UV Transilluminator
• Power Supply (15 W)

Procedure

1. Mix 0.8g agarose with 100 mL 0.5x TAE buffer.
2. Heat by microwave for 1 min 45 sec at ~15-30 sec intervals.
3. Pour the agarose solution into a gel tray with the well comb in place.
4. Keep newly poured gel at room temperature for 20-30 min.
5. Aliquot 1 µL of DNA samples and add 4 µL ddH2O and 1 µL 6x DNA Loading Buffer.
6. Vortex and centrifuge.
7. Remove the comb once the gel is solidified and pour the rest of 1x TBE (75 mL into gel box). Make sure the gel is fully immersed.
8. Load samples into lanes, flanked by 6 µL DNA Ladder.
9. Ensure power to the gel box is turned on. (The side where the DNA is placed is negatively charged - black- and the opposite side is positive - red).
10. Run at 120 V for 40-60 min.

Materials

• Nanodrop Machine
• Elution Buffer
• Wash Buffer
• PCR Product (DNA fragment binding buffer to sample 5:1)

Procedure

1. Nanodrop
2. Select "Nucleotide" on the machine.
3. Make sure the reading is 0 ng/µL (or a negative value) meaning that the machine is clean. If not, grab a kimwipe, wipe the pedestal (top and bottom) and add 2ul of water on the pedestal.
4. Click on "Measure." Once you see the 0 ng/µL reading, proceed to wipe the water from the pedestal.
5. Add 2µL of Qiagen Elution Buffer to the pedestal, this will be your "Blank." Once you see the 0ng/µL from your blank, wipe the liquid off the pedestal. Now you are ready to measure your samples
6. Load 2µL of your sample on the pedestal, click "Measure" and write down the ng/ul value. Don't forget to use a kimwipe to clean the pedestal after measuring each sample.
7. Finally, when you are done using the nanodrop machine, load 2µL of water, click "Measure" and leave a kimwipe on the pedestal so that the next person using it will know it is clean.
8. DNA should have a 260/280 ratio between 1.8-2.0. RNA should be above 2.0. Contaminated DNA will have a low 260/280 of below 1.8.
9. DNA Elution
10. In a 1.5mL microcentrifuge tube, add 2- 7 volumes of DNA binding buffer to each volume of DNA sample(Mix briefly by vortexing).
11. Transfer mixture to a Zymo-Spin column in a collection tube.
12. Centrifuge for 30 seconds. Discard the flow through.
13. Add 200µL DNA Wash Buffer to the column. Centrifuge for 30 seconds. Repeat the wash step.
14. Add >= 6µL DNA Elution Buffer directly to the column matrix and incubate at room temperature for one minute. Transfer the column for 30 seconds to elute the DNA.

Materials

• Thin-walled 200 µL PCR tubes
• Thermocycler

Procedure

1. In PCR tubes, add all the reagents listed in the table above.
2. Briefly spin down the PCR tubes (2-3 s) using a tabletop microcentrifuge in order to ensure that all of the reagents are in the reaction mixture
3. Place the PCR tubes into the selected thermocycler.

4. Once the lid to the thermocycler is properly closed, start the required amplification program, as in the table above.
5. If the amplified DNA is NOT to be immediately used, label the tubes with your initials and date and temporarily store at 4°C or freeze at -20°C for long-term storage in assigned storage box

Materials

• 1x Running Buffer
• 950 mL water
• 50 mL MOPS SDS Running Buffer
• NuPAGE LDS Sample Buffer (4x)
• NuPAGE Reducing Agent (opt.)
• Deionized Water
• Protein Sample
• 8-15 BIS-TRIS gels from 4 C
• Coomassie Blue Stain

Procedure

1. Preparing the Samples
2. Prepare samples according to the table below.
3. Heat the samples at 70 C for 10 minutes.
4. Preparing the Buffers
5. Add 50 mL of 20X NuPAGETM MES or MOPS SDS Running Buffer to 950 mL of deionized water to prepare 1X SDS Running Buffer.
6. For reduced samples, add 1 mL of NuPAGETM Antioxidant to 400 mL 1X SDS Running Buffer.
7. To prepare the gel, remove the comb, and rinse the gel wells three times using 1X Running Buffer.
8. Remove the white tape near the bottom of the gel cassettes.
9. Place the gels in the mini gel tank.
10. Loading the buffers
11. Mini Tank: Add 400 mL of buffer to each chamber.
12. XCell SureLockTM Mini-Cell: Add 600 mL of buffer to the lower chamber, and 200 mL to the upper chamber (for reduced samples, use a running buffer with antioxidants in the upper chamber).
13. Loading the Samples and Ladders
14. Load the appropriate volume of the samples in the appropriate wells.
15. Load the protein ladder in the appropriate well.
16. Running the gel
17. Optimal run times vary depending on gel percentage and power supply used for electrophoresis. If using MES Running Buffer, run for 35 minutes at 200 V constant. If using MOPS Running Buffer, run for 50 minutes at 200 V constant. (If not using a Thermo Fisher Scientific power supply, install Novex Power Supply Adapters.)