Difference between revisions of "Team:NCTU Formosa/Protocol"
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<div class="subtitle"> | <div class="subtitle"> | ||
<h6>Experiment:</h6> | <h6>Experiment:</h6> | ||
| − | <h5>- | + | <h5>- Dual culture technique </h5> |
</div> | </div> | ||
<div class="exp2"> | <div class="exp2"> | ||
<div class="show2"> | <div class="show2"> | ||
<h1>Purpose:</h1> | <h1>Purpose:</h1> | ||
| − | <p> To test | + | <p> To test the antifungal activity of peptides comparing to negative control.</p> |
<div><img class="show2_pic" src="https://static.igem.org/mediawiki/2017/4/4f/Ptp_hide.png" style="display:block; margin:auto;"></div> | <div><img class="show2_pic" src="https://static.igem.org/mediawiki/2017/4/4f/Ptp_hide.png" style="display:block; margin:auto;"></div> | ||
</div> | </div> | ||
<div class="hide2"> | <div class="hide2"> | ||
<h1>Purpose:</h1> | <h1>Purpose:</h1> | ||
| − | <p> To | + | <p> To test the antifungal activity of peptides comparing to negative control. </p> |
<h1>Drugs and equipment:</h1> | <h1>Drugs and equipment:</h1> | ||
<ul> | <ul> | ||
| − | <li> | + | <li>Drugs (ex: NaN<sub>3</sub>, peptides…)</li> |
| − | <li> | + | <li>Hole puncher (tips are recommended)</li> |
| + | <li>PDA (Potato dextrose agar) plate</li> | ||
| + | <li>Fungi plates</li> | ||
| + | <li>Parafilm</li> | ||
<li>Alcohol burner</li> | <li>Alcohol burner</li> | ||
| − | <li> | + | <li>Tweezers</li> |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
</ul> | </ul> | ||
<h1>Process:</h1> | <h1>Process:</h1> | ||
<ol> | <ol> | ||
| − | <li> | + | <li>Culture some plates of fungi. </li> |
| − | <li> | + | <li>Remove all the drugs and tools inside the Hood, disinfect tools with an alcohol burner. You may take a PDA plate for cooling tools down.</li> |
| − | <li> | + | <li>Pick up fungi on the outer cycle (peripheral part) of a plate (the latest part of fungi plate) with tip. Dig into the previous PDA plate and pick up a piece of agar with fungi, remove it onto a new PDA plate, put the piece in the center of the plate.</li> |
| − | <li>Disinfect | + | <li>Disinfect tools with an alcohol burner. Seal up the plate with Parafilm, and label on the name of fungi, date and so on. </li> |
| − | <li>Remove the | + | <li>Remove all the drugs and tools inside the Hood, disinfect tools with an alcohol burner. You may take a PDA plate for cooling tools down.</li> |
| − | <li> | + | <li>Dig holes on the plate with tweezers, the position of the holes should be 0.5cm far away along the radius from the position of latest mycelium as the picture shows.</li> |
| − | + | <li>Introduce the testing drug into the hole (different concentration of peptides, HEPES for our experiments)</li> | |
| − | <li> | + | <li>Disinfect tools with alcohol burner. Seal up the plate with Parafilm.</li> |
| − | <li> | + | <li>Observe how the mycelium grow</li> |
| − | + | <p>這裡有圖</p> | |
</ol> | </ol> | ||
<div><img class="hide2_pic" src="https://static.igem.org/mediawiki/2017/c/cb/Ptp_show.png" style="display:block; margin:auto;"></div> | <div><img class="hide2_pic" src="https://static.igem.org/mediawiki/2017/c/cb/Ptp_show.png" style="display:block; margin:auto;"></div> | ||
| Line 338: | Line 336: | ||
<div class="show3"> | <div class="show3"> | ||
<h1>Purpose:</h1> | <h1>Purpose:</h1> | ||
| − | <p> To test the concentration of spore suspension | + | <p> To test the concentration of spore suspension and calculate germination rate.</p> |
<div><img class="show3_pic" src="https://static.igem.org/mediawiki/2017/4/4f/Ptp_hide.png" style="display:block; margin:auto;"></div> | <div><img class="show3_pic" src="https://static.igem.org/mediawiki/2017/4/4f/Ptp_hide.png" style="display:block; margin:auto;"></div> | ||
</div> | </div> | ||
<div class="hide3"> | <div class="hide3"> | ||
<h1>Purpose:</h1> | <h1>Purpose:</h1> | ||
| − | <p> To test the concentration of spore suspension | + | <p> To test the concentration of spore suspension and calculate germination rate.</p> |
<h1>Drugs and equipment:</h1> | <h1>Drugs and equipment:</h1> | ||
<ul> | <ul> | ||
| Line 370: | Line 368: | ||
<li>(It is recommended to experiment in a laminar flow (hood). Add ddH2O to the plate until water covers the surface of the plate.</li> | <li>(It is recommended to experiment in a laminar flow (hood). Add ddH2O to the plate until water covers the surface of the plate.</li> | ||
<li>Disinfect the glass cell spreaders with an alcohol burner, after cooling down, scrape the plate softly so the spore would be in the water.</li> | <li>Disinfect the glass cell spreaders with an alcohol burner, after cooling down, scrape the plate softly so the spore would be in the water.</li> | ||
| − | <li>Remove the water in the plate to a beaker (You may filter out impurity with gauze). Now you got a spore suspension | + | <li>Remove the water in the plate to a beaker (You may filter out impurity with gauze). Now you got a spore suspension with unknown concentration.</li> |
| − | <li>Clean the Hemocytometer with 75%alcohol and wipe it clean with lens cleaning paper, so as not to make any scratch on it. Put the coverslip on the Hemocytometer, and inject 10ml spore suspension | + | <li>Clean the Hemocytometer with 75%alcohol and wipe it clean with lens cleaning paper, so as not to make any scratch on it. Put the coverslip on the Hemocytometer, and inject 10ml spore suspension from the tiny chamber beside. |
The spore suspension should cover all the square of the Hemocytometer. </li> | The spore suspension should cover all the square of the Hemocytometer. </li> | ||
<li>Put the Hemocytometer under a microscope, and observe the spore.</li> | <li>Put the Hemocytometer under a microscope, and observe the spore.</li> | ||
| − | <li>Count the amount of the spore in the square, and calculate the concentration. Add water if it’s concentration is too high; centrifuge the liquid if the concentration is too low. Finally, you got a spore suspension | + | <li>Count the amount of the spore in the square, and calculate the concentration. Add water if it’s concentration is too high; centrifuge the liquid if the concentration is too low. Finally, you got a spore suspension with concentration |
| − | you know. We would like to prepare spore suspension | + | you know. We would like to prepare spore suspension with the concentration of 1.5*10^5 spores/ml for our experiments.</li> |
| − | <li>Mixed 5ul spore suspension | + | <li>Mixed 5ul spore suspension with 5ul 2% glucose solution and 5ul peptide together into a PCR tube (It is recommended for pipetting 50 times to ensure that there is a mix of uniform).</li> |
<li>Draw 15μl of the solution from PCR tube to the double concave slide, and put the slide into a Petri dish. Add some ddH2O around the slide, in order to create an environment of 100% relative humidity so that the liquid would note evaporate | <li>Draw 15μl of the solution from PCR tube to the double concave slide, and put the slide into a Petri dish. Add some ddH2O around the slide, in order to create an environment of 100% relative humidity so that the liquid would note evaporate | ||
easily.</li> | easily.</li> | ||
| Line 414: | Line 412: | ||
<h1>Process:</h1> | <h1>Process:</h1> | ||
<ol> | <ol> | ||
| − | <li>Soak the plant tissue to sterilization: All parts of | + | <li>Soak the plant tissue to sterilization: All parts of plant should soak in alcohol for 1 minute. For leaves, skim over NaClO for a few times; for roots, soak in NaClO for 1 minute; for mature stem parts, soak in NaClO for 20~30 |
seconds; for tender stems, skim over NaClO for a few times. All of these parts should be washed with ddH 2 O.</li> | seconds; for tender stems, skim over NaClO for a few times. All of these parts should be washed with ddH 2 O.</li> | ||
| − | <li>Remove all the drugs and tools inside the Hood, disinfect | + | <li>Remove all the drugs and tools inside the Hood, disinfect knifes and tweezers with alcohol burner. You may take a PDA plate for cool down.</li> |
| − | <li>Cut the infected plant tissue for an area of 5mm 2, remove them all to new PDA plates. The side with hypha should put downward, and be towed on the plate. The amount of tissue which | + | <li>Cut the infected plant tissue for an area of 5mm 2 , remove them all to new PDA plates. The side with hypha should put downward, and be towed on the plate. The amount of tissue which are put on one single plate depends on the range |
of cutting area.</li> | of cutting area.</li> | ||
<li>Seal up the plate with Parafilm, and label on the name of fungi, date and so on.</li> | <li>Seal up the plate with Parafilm, and label on the name of fungi, date and so on.</li> | ||
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Revision as of 14:54, 1 November 2017
Wet Lab Protocol
Experiment:
- Preparation for Potato dextrose agar (PDA) plates
Purpose:
To prepare solid medium to civilize the fungi
Purpose:
To prepare solid medium to civilize the fungi
Drugs and equipment:
- PDA powder
- ddH2O
- Petri dishes
- Serum bottle
- Microwave oven
- Autoclave
- Alcohol burner
Process:
- The ratio of PDA powder and ddH2O is 39g : 1000ml. To prevent the liquid spill out from Serum bottles, we usually have only 300ml of the liquid in a 500ml Serum bottle.
- Loosen the bottle cap for a bit, and put it into the autoclave.
- Screw the bottle cap tight to cool down. If the liquid becomes solid, heat it up with a microwave oven.
- Bring the bottle into the Hood, disinfect bottleneck with alcohol burner. Pour out the liquid to plates, each plate can hold 15~20ml of the PDA liquid. Shake the plate smoothly and make the liquid surface flat.
- Keep the plates in normal temperature or in 4 °C refrigerator
Experiment:
- Dual culture technique
Purpose:
To test the antifungal activity of peptides comparing to negative control.
Purpose:
To test the antifungal activity of peptides comparing to negative control.
Drugs and equipment:
- Drugs (ex: NaN3, peptides…)
- Hole puncher (tips are recommended)
- PDA (Potato dextrose agar) plate
- Fungi plates
- Parafilm
- Alcohol burner
- Tweezers
Process:
- Culture some plates of fungi.
- Remove all the drugs and tools inside the Hood, disinfect tools with an alcohol burner. You may take a PDA plate for cooling tools down.
- Pick up fungi on the outer cycle (peripheral part) of a plate (the latest part of fungi plate) with tip. Dig into the previous PDA plate and pick up a piece of agar with fungi, remove it onto a new PDA plate, put the piece in the center of the plate.
- Disinfect tools with an alcohol burner. Seal up the plate with Parafilm, and label on the name of fungi, date and so on.
- Remove all the drugs and tools inside the Hood, disinfect tools with an alcohol burner. You may take a PDA plate for cooling tools down.
- Dig holes on the plate with tweezers, the position of the holes should be 0.5cm far away along the radius from the position of latest mycelium as the picture shows.
- Introduce the testing drug into the hole (different concentration of peptides, HEPES for our experiments)
- Disinfect tools with alcohol burner. Seal up the plate with Parafilm.
- Observe how the mycelium grow
這裡有圖
Experiment:
- Spore germination percentage
Purpose:
To test the concentration of spore suspension and calculate germination rate.
Purpose:
To test the concentration of spore suspension and calculate germination rate.
Drugs and equipment:
- 75% Alcohol
- 2% glucose solution
- ddH2O
- HEPES buffer
- Peptides
- Alcohol burner
- Hemocytometer
- Fungi plates
- Glass Cell Spreaders
- 10ul pipet
- Gauze
- Centrifuge
- Beaker*2
- Petri dishes
- Lens cleaning paper
- Microscope
- Slide glass with 2 Cavities
- Counter
Process:
- Choose fungi plates that have produced spores.
- (It is recommended to experiment in a laminar flow (hood). Add ddH2O to the plate until water covers the surface of the plate.
- Disinfect the glass cell spreaders with an alcohol burner, after cooling down, scrape the plate softly so the spore would be in the water.
- Remove the water in the plate to a beaker (You may filter out impurity with gauze). Now you got a spore suspension with unknown concentration.
- Clean the Hemocytometer with 75%alcohol and wipe it clean with lens cleaning paper, so as not to make any scratch on it. Put the coverslip on the Hemocytometer, and inject 10ml spore suspension from the tiny chamber beside. The spore suspension should cover all the square of the Hemocytometer.
- Put the Hemocytometer under a microscope, and observe the spore.
- Count the amount of the spore in the square, and calculate the concentration. Add water if it’s concentration is too high; centrifuge the liquid if the concentration is too low. Finally, you got a spore suspension with concentration you know. We would like to prepare spore suspension with the concentration of 1.5*10^5 spores/ml for our experiments.
- Mixed 5ul spore suspension with 5ul 2% glucose solution and 5ul peptide together into a PCR tube (It is recommended for pipetting 50 times to ensure that there is a mix of uniform).
- Draw 15μl of the solution from PCR tube to the double concave slide, and put the slide into a Petri dish. Add some ddH2O around the slide, in order to create an environment of 100% relative humidity so that the liquid would note evaporate easily.
- After incubating under 20℃ for 6 hours, we observe and classify the spores into four grades according to the length of the germination tube, calculate the percentage of each germination grade of each sample.
這裡有圖
這裡有圖
Experiment:
- Separation of infected plant tissue
Purpose:
To separate infected plant tissue and cultivate pathogen fungi
Purpose:
To separate infected plant tissue and cultivate pathogen fungi
Drugs and equipment:
- 75% Alcohol
- 0.5%~1% Sodium hypochlorite (NaClO)
- ddH2O
- PDA (Potato dextrose agar) plate
- Parafilm
- Alcohol burner
- Plants
- Knifes
- Tweezers
Process:
- Soak the plant tissue to sterilization: All parts of plant should soak in alcohol for 1 minute. For leaves, skim over NaClO for a few times; for roots, soak in NaClO for 1 minute; for mature stem parts, soak in NaClO for 20~30 seconds; for tender stems, skim over NaClO for a few times. All of these parts should be washed with ddH 2 O.
- Remove all the drugs and tools inside the Hood, disinfect knifes and tweezers with alcohol burner. You may take a PDA plate for cool down.
- Cut the infected plant tissue for an area of 5mm 2 , remove them all to new PDA plates. The side with hypha should put downward, and be towed on the plate. The amount of tissue which are put on one single plate depends on the range of cutting area.
- Seal up the plate with Parafilm, and label on the name of fungi, date and so on.
- Put the plate in the best environment to observe if the fungi or other species of microorganism grow.
