RNAi Experiments

Inoculating LB+ampicillin Liquid Cultures with Human cDNA Library Clones

1. Prepare a solution of LB + 100 mg/ml ampicillin in a sterile 50-ml freestanding tube using LB broth and 100 mg/ml ampicillin stock solution (stored in freezer or refrigerator). For every human cDNA library clone that you test you will need 1 ml of LB + 100 mg/ml ampicillin solution. Each school is expected to screen 30 random clones. You may screen as many clones at a given time as you wish. For example, you may want to screen 15 clones per week for two weeks. You will have to decide the pace at which you screen the library.

2. To sterile snap-cap tubes transfer 1 ml of LB + 100 mg/ml ampicillin using a 10-ml sterile pipet.

3. Label your LB + amp-containing snap-cap tubes with unique sample numbers (for example, you can start at 1 and go up.)

4. Inoculate each tube with a single bacterial colony from the Human cDNA Library plate by touching a sterile wooden stick to the colony (making certain that some of the cells have been transferred to the stick) and then dipping the stick into the liquid and shaking it a bit.

5. Also inoculate a tube with a single colony from the pos-1 plate (this will serve as your positive control) and inoculate a tube with a single colony from the TriplEx plate (this will serve as your negative control). Be sure to label your control culture tubes appropriately.

6. Let the liquid culture grow at room temperature for 2 days. Just place the tube in a test tube rack and let it sit on the bench at room temperature (it does not need to be shaken). After two days tap or gently vortex your human cDNA library liquid cultures to resuspend any cells that may have settled. The culture should be very cloudy with bacterial growth. You will need this liquid culture to seed IPTG worm plates.

Seeding IPTG Worm Plates with Library Clone Cultures

1. For each liquid culture take one IPTG Worm plates out of the refrigerator and put it on the bench for at least 1 hr. to allow it to warm to room temperature.

2. Tap or gently vortex your cultures to resuspend any cells that have settled to the bottom of the culture tube. Using a P-200 and sterile pipeman tips, transfer 90 ml of each culture to an appropriately labeled IPTG worm plate. Let the plate sit upright until the 90 ml spot drys (this may take overnight).

Transferring Worms to Seeded Plates

1. Using a burner, flame the end of the worm pick to sterilize it. Wave the pick in the air for a few seconds to allow it to cool down. (Alternately, sterilize the end of the worm pick by inserting it into a small container of 95% ethanol, then wave it for a few seconds to allow the ethanol to evaporate.) Place a plate of lin-2 worms under the microscope.

2. Transfer 5 medium sized worms to each seeded IPTG Worm plate. After every few worms it is best to re-sterilize your worm pick.

3. Store your worm-containing plates upside-down on the bench at room temperature. Be prepared to check your worms the next day.

Analyzing your RNAi Results

The day after you added worms to your seeded plate you should look at your worm plates under the microscope. You may not see any bag of worms or bag of eggs yet. If this is the case, examine your plates on Day 2 (as well as Day 3) and answer the following questions:

1. How many large adult nematodes are there? You will have to ignore smaller progeny and eggs when you are counting.

2. How many old adults have the bag-of-worms phenotype?

3. How many old adults appear to be bags with unhatched eggs inside?

4. How many old adults appear to be bags of dead eggs?

5. Does the control plates appear as expected? Pos-1, a positive control gene that is essential for embryogenesis, should be bags of eggs. TriplEx, a negative control culture, should have no effect on embryogenesis and is expected to have all animals as bags of worms.

6. What can you conclude about the role of your human library gene in early development? Why?

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