Difference between revisions of "Biemar:EmbryoCollection"
(New page: GENERAL GUIDELINES FOR COLLECTING DROSOPHILA EMBRYOS The secret to obtaining large quantities of embryos is to keep your flies happy. Maintaining happy flies is best achieved by observin...)
Revision as of 07:13, 8 July 2010
GENERAL GUIDELINES FOR COLLECTING DROSOPHILA EMBRYOS
The secret to obtaining large quantities of embryos is to keep your flies happy. Maintaining happy flies is best achieved by observing a few simple rules. First, flies that have experienced the collection chamber for 2-3 days, with frequent feeding will generally start producing eggs full blast. I typically set-up collection chambers on a Friday afternoon and feed them regularly over the week-end so that, on the following Monday, they're good to go. Second, flies will tend to lay poorly on AJ plates that are too cold. I try to take the plates I will need out of the fridge at least two hr prior to use. Third, flies are happier in clean collection chambers, thus I change them every morning. There is another reason to change collection chambers every day; flies won't lay their eggs exclusively on the AJ plate and they also tend to lay eggs where others are already present. The more your flies lay their eggs anywhere else than the AJ plate, the fewer embryos you can collect... obviously.
● Apple juice plates: - Stir 24 gr of Bacto Agar into ~ 650 ml of dH2O and microwave 8-10 min. or until the solution develops froth. Be careful not to let it boil over. It's done when there is no more Bacto Agar visible. - Bring the solution to 1 L with Apple juice and stir. - Pour onto plates about half full, using a 60 ml syringe. Be careful not to overfill. When bubbles appear, quickly suck them back into the syringe. - Line a Tupperware® container with moistened paper towels and stack the plates sideways (like Oreos) and store at 4ºC.
● Yeast paste: - Mix dry yeast with dH2O until it forms a nice homogenous paste that has the consistency of peanut butter. - Fill only 1/3 of the recipient and let settle at RT for a few hr to let the yeast “expand”. Cover the recipient to avoid flies contaminating it. Store at 4ºC.
As in other model organisms, embryos are “staged” according to morphological landmarks (see “The Embryonic Development of Drosophilae Melanogaster” by Jose Campos-Ortega and Volker Hartenstein. ). In Drosophila, these are referred to in terms of hr after egg laying (h AEL). One typically collects embryos in 1-3 hr. time intervals (e.g. 2-4h, 3-6h, 15-16h,...). We achieve this by letting females lay their eggs for a certain amount of time – the 1-3 hr. time window just mentioned – and then “aging” the embryos for the amount of time required to attain the correct stage. Thus, if one want to look at “15-16 hAEL” embryos, one will collect AJ plates every hr and let each plate “age” for an additional 15 hr before fixing the embryos.
Example of a general scheme to get 2-4 hrs old embryos - 9am: replace the o/n plate with a new one and place the o/n plate at 18°C. - 11am: replace the 9am plate with a new one and place the 9am plate at 18°C. - 1pm: replace the 11am plate with a new one and place the 11am plate at18°C. - 3pm: replace the 1pm plate by new one and keep 1pm plate at RT. Collect embryos from the o/n and 9am plates. - 5pm: replace the 3pm plate by a new one (for o/n collection) and place the 5pm plate at 18ºC (mix this one with the next overnight collection). Collect embryos from the 11am and 1pm plates.