Nematode dye filling
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Nematode dye filling
(Ed Hedgecock [modified by M. Herman/K. Williams/S.M. Hettenbach])
Thumb plate
- Transfer worms into a known amount of M9 in a labeled depression.
- Add DiO(2 mg/ml in DMF) at 10 l/ml of M9.
- Let sit for 2 hours.
- Transfer worms with a Pasteur pipet in as little liquid as possible to a feeding plate.
- Allow the plates to sit at room temperature (RT) or at 20°C for 30 minutes to 2 hours to pass the dye out of the digestive tract. Worms dyed late in the afternoon can be stored at 15°C overnight (o/n) and viewed the next morning.
- Label 1.7 ml microfuge tube with strain name.
- Squirt ~1 ml M9 onto plate with a pasteur pipet. Rinse plate with M9 using a pasteur pipet and transfer solution to labeled microfuge tube.
- Spin for 10-20 seconds in a mini centrifuge and carefully remove supernatant (s/n). Worm pellet will be very loose.
- Wash once with M9. Optional -- don't do if pellet is very small.
- Add 1 ml M9.
- Add DiO (2 mg/ml in DMF) at 5 l/ml of M9.
- Rock or rotate the tubes for 2 hours at RT or 20°C.
- Spin for 10-20 seconds in the mini centrifuge and carefully remove s/n. Worm pellet will be very loose.
- Wash once with M9. NOT optional.
- Allow the plates to sit at RT or at 20°C for 30 minutes to 2 hours to pass the dye out of the digestive tract. Worms dyed late in the afternoon can be stored at 15°C o/n and viewed the next morning.
- Label 15 ml centrifuge (c/f) tube with strain name.
- Squirt ~1-2 ml M9 onto plate with a pasteur pipet. Rinse plate with M9 using a pasteur pipet and transfer solution to c/f tube.
- Spin pulse on high in a clinical c/f and carefully remove s/n. Worm pellet will be very loose.
- Wash once with M9.
- Add 2 ml M9.
- Add DiO (2 mg/ml in DMF) at 5 l/ml of M9.
- Rock the tubes for 2 hours at RT on a lab quake rocker.
- Spin pulse on high in a clinical c/f and carefully remove s/n. Worm pellet will be very loose.
- Wash once with M9.
- Transfer worm pellet to labeled plate(s). Use enough plates that worms have sufficient food.
- Allow the plates to sit at RT or at 20°C for 10 minutes to 2 hours to pass the dye out of the digestive tract. Worms dyed late in the afternoon can be stored at 15°C o/n and viewed the next morning.
Big Scope
- Make pads using 5% agar with 10mM NaN3 (10 l of 1M NaN3 /ml of 5% agar -- our test tubes contain 4 ml of 5% agar).
- Apply 4-6 l of M9 to one end of the pad.
- Transfer 25-50 worms to the M9, adding more M9 if it starts to evaporate.
- Put cover slip on and view with upright and fluorescence.
- View worms on feeding plate using dissecting microscope.
- Use white light on lowest setting.
- Adjust intensity of white light using mirror. Do not turn lamp source on and off.