Cage set up for Domeless fly egg laying, then egg collection and cell culture preparation the day after. Trying using trypsin to break up cell clumps.

2021 10 11 Yeast Pate on Apple Juice Plates

  • Paced two streaks of yeast on two apple juice plates, covered, and let sit overnight
    • Yeast is the same made last week

2021 10 12 Breeding Cage Setup

  • Using two vials from 9/20 that are very full of flies (other times, 3 vials are needed)
  • Washed the cage with tap water, paper towel, distilled water, then paper towel again
  • Took plate and cage to fly room
  • Plugged in CO2 and turned on valve
  • Rotated and pulled out the cotton plug on the first vial
  • Tipped the vial over onto the CO2 plate
  • Kept tapping until ~all the flies had come down, moved the vial around the CO2 plate to not squish the flies
  • Used a paint brush to move the files to a corner
  • Repeated process with the second vial
  • Plugged and tossed the old vials in the fly trash
  • Put the yeast plate on the red section of the cage
  • Brushed the flies gently into the clear cage
  • Put sideways and put the red cap on the cage
  • Rotated the cage slowly on it’s side on the bench for ~5 minutes so the flies could wake up
  • Set the cage upright on the bench for overnight
  • Turned off and unplugged the CO2

2021 10 13 Domeless Egg Collection and Cell Culture Prep

  • Made 50% wash solution
    • 10mL bleach
    • 10mL 1X wash
  • Turned on UV and blower in the TC hood for 15 minutes
  • Took out 0.25% trypsin and medium from the fridge to warm on the bench then into the hood after the UV stopped

Fly Room

  • Set up CO2
  • Tapped fly cage onto the CO2 plate until all flies were asleep
  • Took out red cap and plate and covered the plate immediately. Plate had a lot of dead flies and a lot of eggs
  • Put in new yeast plate to the red part and re-capped cage. _accidentally pushed my nail into the agar while setting up
  • Rotated the cage horizontally until the flies woke up
  • Set up the cage for overnight
  • Turned off all CO2

Filtering

  • Made small beaker with 70& ethanol
  • Picked off dead flies and put in ethanol
  • Set up the filter rig: autoclaved erlenmeyer flask, 100um filter, 400um filter, then the funnel
  • Squirted water into the plate and began mixing up the yeast with a paint brush
  • Tipped over the plate into the funnel and squirted water to wash the liquid down
  • Not all the yeast was gone so I brushed and rinsed two more times
  • Took off the funnel and rinsed the green filter with water
  • Took of the green filter and rinsed the yellow with 1X wash for close to 2 minutes. There was some black flakes that made it onto the yellow filter, probably fly parts
  • Flipped the yellow filter over into a new 50mL conical
  • Serologically pipetted 10mL 50% bleach into the filter to wash out the eggs
  • Used a pasteur pipette to transfer the 10mL to a siliconized 10mL tube
  • Let sit for 3 minutes: lots of eggs settled!
  • Centrifuged for 3 minutes at 400rcf

In Tissue Culture Hood

  • Made sure trypsin was at room temp
  • Turned on the vacuum pretty light
  • Used siliconized pasteur pipette (SPP) and the vacuum to aspirate off the bleach
  • Added 10mL trypsin to the 10mL tube and inverted the liquid turned purple! The red dye in the trypsin turns purple with basic pH
  • Centrifuged 3 min 400rcf
  • Asprirated off the liquid with an SPP
  • Added 10mL trypsin and inverted the liquid was less purple this time
  • Centrifuged 3 min 400rcf
  • Asprirated off the liquid with an SPP
  • Added 10mL trypsin and inverted the liquid was pretty close to the usual red
  • Centrifuged 3 min 400rcf
  • Asprirated off the liquid with an SPP to about ~200ul
  • Added back in 2mL fresh trypsin
  • Unwrapped dounce mortar and put in a separate rack
  • Used the SPP to pipette mix once and transfer all the liquid from the 10mL tube to the dounce
  • Homogenized for ~30 seconds, press down and twist, then release up, about 3 times
  • Laid the pestle back down on the clean autoclaved foil
  • Let the dounce with the sample sit for 30 min in the hood
    • Every ~10 minutes, took the pestle and put it in the dounce and lifted up (no pressure) once just to mix the liquid
  • After the incubation: used an SPP to transfer the liquid from the dounce into a new siliconized 10mL glass tube (the old tube had un-homogenized eggs stuck to the sides)
  • Added 8mL 420 medium without serum to the 10mL tube and inverted
  • Centrifuged 800rcf for 3 minutes (program 1)
  • Aspirated off the liquid with an SPP
  • Added 10mL 420 medium without serum and inverted
  • Centrifuged 800rcf for 3 minutes (program 1)
  • Aspirated off the liquid with an SPP
  • Added 2mL medium with serum to the 10mL tube
  • Made two new 25cm2 flasks
    • 10263 domeless P1 primary 10/13 MES
  • Added 8mL 420 medium with serum to one flask
  • Used SPP and bulb to transfer the liquid from the 10mL tube to the flask with the medium
  • Used a 5mL serological pipette to pipette mix once, then transfer 5mL to the second flask
  • Laid flasks flat and placed in the 23 degree incubator
  • Cleaned up:
    • Rinsed: flask, dounce, beaker, and 10mL tubes
    • Add 1 drop of detergent to the 10mL tube and rinse: removes all the stuck eggs!
    • Cleared away pipettes, wiped work station, autoclaved with rinsed items