MattPiperLab: set up Drosophila lifespan experiment: Difference between revisions
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== materials == | == materials == | ||
'''purps''' | '''purps (egg laying plates)''' | ||
* petri dishes | * petri dishes | ||
* grape juice | * grape juice | ||
* agar | * agar | ||
--> ensure sufficient agar in mix to yield a hard surface that won't be disturbed when washing off eggs | |||
'''bottles''' | '''bottles''' | ||
* half-pint milk bottles | * half-pint milk bottles | ||
* 70ml 1xSY food | * 70ml 1xSY food | ||
(see Bass et al, 2007, J Gerontol, 62A,1071-81 for food making supplies and method) | |||
'''1xSY food''' | '''1xSY food''' | ||
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'''Parental generation''' | '''Parental generation''' | ||
* purp population cages (for best results, purp cage twice over 48h and use the second purp for egg squirts) | * put purp in population cages overnight (for best results, purp cage twice over 48h and use the second purp for egg squirts) | ||
* wash eggs from purps with PBS into falcon tube and allow to settle | * wash eggs from purps with PBS (dislodging with a paint brush) into falcon tube and allow to settle | ||
* using a wide-bore tip (or cut off yellow tip 6mm from bottom) and a 100ul pipette, squirt eggs into 1.0xSY bottles (20ul per bottle should give about 250 – 300 flies per bottle) | * using a wide-bore tip (or cut off yellow tip 6mm from bottom) and a 100ul pipette, squirt eggs into 1.0xSY bottles (20ul per bottle should give about 250 – 300 flies per bottle) | ||
(note that to uptake a densely-packed tip of eggs, rapidly release the plunger on the pipette and drop the tip further into the egg mass) | (note that to uptake a densely-packed tip of eggs, rapidly release the plunger on the pipette and drop the tip further into the egg mass) |
Latest revision as of 11:47, 21 January 2015
materials
purps (egg laying plates)
- petri dishes
- grape juice
- agar
--> ensure sufficient agar in mix to yield a hard surface that won't be disturbed when washing off eggs
bottles
- half-pint milk bottles
- 70ml 1xSY food
(see Bass et al, 2007, J Gerontol, 62A,1071-81 for food making supplies and method)
1xSY food per litre:
- 15g agar (Sigma)
- 50g sucrose (Tate & Lyle)
- 100g lyophilised yeast (MPBIo brewer's yeast)
- 30ml nipagin solution (clariant UK Ltd) - (100 g/L methyl 4-hydroxybenzoate in 95% ethanol)
- 3ml propionic acid (Sigma)
Standard density fly rearing
Parental generation
- put purp in population cages overnight (for best results, purp cage twice over 48h and use the second purp for egg squirts)
- wash eggs from purps with PBS (dislodging with a paint brush) into falcon tube and allow to settle
- using a wide-bore tip (or cut off yellow tip 6mm from bottom) and a 100ul pipette, squirt eggs into 1.0xSY bottles (20ul per bottle should give about 250 – 300 flies per bottle)
(note that to uptake a densely-packed tip of eggs, rapidly release the plunger on the pipette and drop the tip further into the egg mass)
- on 10th day, transfer all flies that have emerged to fresh bottles and maintain tipping every-other-day until ready to set up cage for experimental generation
experimental generation
- put 4 to 5 bottles of parental flies into cage (1l yoghurt container cage)
- purp cages over 48h with live yeast paste, using the eggs from the overnight lay on the second purp for experimental flies
- wash eggs from purp with PBS into falcon tube and allow to settle
- cut off yellow tip and squirt 20ul eggs per bottle
- on the 9th day (evening) clear bottles
- on the 10th day transfer emerged flies to fresh bottles
- allow to mate for 2 days for once-mated females or male experiments.
Experimental set-up
anaesthetise flies, sex and count into vials or bottles of difft food concentrations. This is day 0 for experimental treatment.
anaesthetising flies
- put needle from gas outlet into a clean, dry bottle and allow to fill with CO2
- tip flies from bottle into this bottle of gas to knock-out, and transfer anaesthetised flies to CO2 pad
- while sexing / counting flies, put needle back in empty bottle to fill with gas again ready for next bottle of flies
allocating flies to treatments
- flies need to be allocated to different treatments systematically to avoid one bottle supplying all the flies to one or a few treatments:
- eg. When working with 5 food treatments (0.1, 0.5, 1.0, 1.5 and 2.0 SY) in vials
* working with anaesthetised flies from one bottle, transfer 10 flies to 0.1, then 0.5, then 1.0, then 1.5, then 2.0 * there should be enough flies from one bottle (~300) to do up to 3 vials per treatment in this case, if there aren’t enough remaining on the pad for another full set of treatments (ie 50 flies) discard remainders and start a new bottle * to control for duration of gassing, when working with the next bottle of anaesthetised flies, first allocate to treatment 0.5, then 1.0, then 1.5, then 2.0 and then 0.1.
example time-line
- Wed: put egg laying plate with live yeast paste into population cage overnight
- Thur: retrieve egg laying plate from population cage. late afternoon replace with a fresh plate with a small amount of live yeast paste
- Fri: in the morning, retrieve egg plate, wash off eggs to collect and perform egg squirts
- Sat:
- Sun:
- Mon:
- Tue:
- Wed:
- Thur:
- Fri:
- Sat:
- Sun:
- Mon: transfer freshly emerged flies into a new bottle (parental generation)
- Tue:
- Wed: gas flies and combine 3-4 bottles into cage with yeast paste on a fresh purp
- Thur: retrieve egg laying plate from population cage. late afternoon replace with a fresh plate with a small amount of live yeast paste
- Fri: in the morning, retrieve egg plate, wash off eggs to collect and perform egg squirts
- Sat:
- Sun:
- Mon:
- Tue:
- Wed:
- Thur:
- Fri:
- Sat:
- Sun:
- Mon: transfer freshly emerged flies into a new bottle
- Tue:
- Wed: gas flies and sort/allocate into vials for treatment (=day 0 of experiment)