Difference between revisions of "UW-Stout/Heat Shock SP21"
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'''Calibration Experiment Materials''': | '''Calibration Experiment Materials''': | ||
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• Distilled H2O | • Distilled H2O | ||
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• Wild-type yeast cells | • Wild-type yeast cells | ||
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• P20 and P200 micro pipettors | • P20 and P200 micro pipettors | ||
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• PCR test tubes (mini) | • PCR test tubes (mini) | ||
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• Glass beads | • Glass beads | ||
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• 15 agar filled petri dishes | • 15 agar filled petri dishes | ||
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1. Vortex the yeast culture briefly to resuspend the yeast pellet. | 1. Vortex the yeast culture briefly to resuspend the yeast pellet. | ||
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2. Set up 5 different PCR test tubes with 50 ul of wild-strain yeast cells and 50 ul of distilled water in each. | 2. Set up 5 different PCR test tubes with 50 ul of wild-strain yeast cells and 50 ul of distilled water in each. | ||
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3. Label each tube “A, B, C, D, E”. | 3. Label each tube “A, B, C, D, E”. | ||
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4. Insert test tube A in the thermocycler for exactly 30 minutes at 48°C. | 4. Insert test tube A in the thermocycler for exactly 30 minutes at 48°C. | ||
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5. After test tube A is done cycling, place test tube B in the thermal cycler for exactly 15 minutes at 48° C. | 5. After test tube A is done cycling, place test tube B in the thermal cycler for exactly 15 minutes at 48° C. | ||
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6. After test tube B is done cycling, place test tube C in the thermal cycler for exactly 30 minutes at 48° C. | 6. After test tube B is done cycling, place test tube C in the thermal cycler for exactly 30 minutes at 48° C. | ||
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7. After test tube C is done cycling, place test tube D in the thermal cycler for exactly 45 minutes at 48° C. | 7. After test tube C is done cycling, place test tube D in the thermal cycler for exactly 45 minutes at 48° C. | ||
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8. After test tube D is done cycling, place test tube E in the thermal cycler for exactly 60 minutes at 48° C. | 8. After test tube D is done cycling, place test tube E in the thermal cycler for exactly 60 minutes at 48° C. | ||
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9. Carry on and analyze/count the DNA colonies in the wells. | 9. Carry on and analyze/count the DNA colonies in the wells. | ||
| − | + | 10. Repeat 3 trials | |
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'''Knockout Experiment Materials''': | '''Knockout Experiment Materials''': | ||
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| + | • Knockout yeast strains | ||
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| + | • Wild-type strain | ||
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| + | • 21 Agar filled petri dishes | ||
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| + | • Distilled water | ||
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| + | • Micro pcr tubes | ||
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| + | • Glass beads | ||
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| + | '''Knockout Procedure''' | ||
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| + | 1. Set thermocycler to 48°C sitting temperature. | ||
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| + | 2. Vortex the yeast culture briefly to resuspend the yeast cells. | ||
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| + | 3. Label 7 pcr tubes A through G. | ||
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| + | 4. Micropipette 50 ul of water into the pcr tubes. | ||
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| + | 5. Micropipette 50 ul of each strain into the tubes according to this table: | ||
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| + | 6. Place pcr tubes into thermocycler for 30 minutes. | ||
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| + | 7. Label each petri dish accordingly. | ||
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| + | 8. Micropipette the strains onto the coordinated petri dish. | ||
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| + | 9. Using 4-5 glass beads, spread the mixture around. | ||
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| + | 10. Place all the petri dishes in the incubator (30°C). | ||
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| + | 11. Let sit for 48 hours. | ||
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| + | 12. Take out and analyze the growth. | ||
| + | |||
| + | 13. Repeat for 3 trials. | ||
| + | [[File:IMG_1610.jpg|center|thumb|500px|Pitri Dish]] | ||
Latest revision as of 10:49, 27 April 2021
Preliminaries: None
Calibration Experiment Materials:
• Distilled H2O
• Wild-type yeast cells
• P20 and P200 micro pipettors
• PCR test tubes (mini)
• Glass beads
• 15 agar filled petri dishes
Calibration Experiment Procedure:
1. Vortex the yeast culture briefly to resuspend the yeast pellet.
2. Set up 5 different PCR test tubes with 50 ul of wild-strain yeast cells and 50 ul of distilled water in each.
3. Label each tube “A, B, C, D, E”.
4. Insert test tube A in the thermocycler for exactly 30 minutes at 48°C.
5. After test tube A is done cycling, place test tube B in the thermal cycler for exactly 15 minutes at 48° C.
6. After test tube B is done cycling, place test tube C in the thermal cycler for exactly 30 minutes at 48° C.
7. After test tube C is done cycling, place test tube D in the thermal cycler for exactly 45 minutes at 48° C.
8. After test tube D is done cycling, place test tube E in the thermal cycler for exactly 60 minutes at 48° C.
9. Carry on and analyze/count the DNA colonies in the wells.
10. Repeat 3 trials
Knockout Experiment Materials:
• Knockout yeast strains
• Wild-type strain
• 21 Agar filled petri dishes
• Distilled water
• Micro pcr tubes
• Glass beads
Knockout Procedure
1. Set thermocycler to 48°C sitting temperature.
2. Vortex the yeast culture briefly to resuspend the yeast cells.
3. Label 7 pcr tubes A through G.
4. Micropipette 50 ul of water into the pcr tubes.
5. Micropipette 50 ul of each strain into the tubes according to this table:
6. Place pcr tubes into thermocycler for 30 minutes.
7. Label each petri dish accordingly.
8. Micropipette the strains onto the coordinated petri dish.
9. Using 4-5 glass beads, spread the mixture around.
10. Place all the petri dishes in the incubator (30°C).
11. Let sit for 48 hours.
12. Take out and analyze the growth.
13. Repeat for 3 trials.
