* Embryos from Cage #1 are always fixed at 7:30pm the same day they are collected
** Embryos from Cage #2 are fixed at three different times depending on the desired interval (0-3, 3-6 at 1:30 pm same day as collected; 6-9,9-12 at 7:30 pm same day as collected; 12-15, 15-18 at 7:30 am the day after collection).
16. Carefully remove any flies that may have stuck to the fly food or the yeast paste to prevent further egg deposition. While removing the flies take care not to damage the deposited embryos.
17. Pour enough deionized H₂O on the tray to cover the entire area. Use paintbrush to collect the embryos from the tray and carefully pour them into the prepared three level sieve. The first sieve (20) will collect pieces of loose agar, the second sieve(40) will collect loose fly parts (e.g. abdomens, wings and heads) and the third sieve will collect the clean embryos.
18. Must be large enough to contain one level of the sieve and high enough so that the embryos in the sieve are can be submerged in liquid. (e.g. 100% bleach, deionized H₂O).
19. Timing of this step is critical. If the embryos are left in bleach for too long they will be irreparably damaged.
20. Heptane must be discarded into a properly labeled heptane waste container.
21. Formaldehyde/PBS mixture must be prepared fresh just prior to use.  Formaldehyde is a classified as a human carcinogen and should be handled with caution and discarded into a properly labeled Formaldehyde waste container.
22. When removing the upper (heptane) phase, start at the interphase and work your way up. Use Falcon Pipetaid.
23. Good embryos, unbroken embryos devoid of chorion and vitelline membranes, will sink to the bottom. Embryos remaining at interphase are damaged and should not be used in hybridization.
24. Methanol must be discarded into a properly labeled methanol waste container.
25. Do not put more than 7 ml of embryos in a 50 ml falcon tube. Lay the falcon tube on its side to prevent crushing the embryos.
26. The embryo master mix is created by combining equal volumes of embryos of all collected stages. It is best to measure out the individual stages in an eppendorf tube and then transfer individual time intervals into a prepared falcon tube. Let the embryos settle to ensure equal volumes. Easiest way to transfer the embryos is by turning the eppendorf tube upside down above the falcon tube and washing out the embryos with methanol using the pipetaid.
27. Waste from steps 3,4 and 5 of the hybridization protocol (3.2.3) must be discarded into a properly labeled Formaldehyde waste container.
28. Use 12ml total (9ml methanol, 2.88ml PBS, 120ul formaldehyde)-prepare fresh just before starting the hybridization steps.
29. Use 12ml total (3ml methanol, 8.64ml PBS, 360ul formaldehyde)- prepare fresh just before starting the hybridization steps.
30. Use 12ml total (11.52ml PBS, 480ul formaldehyde)- prepare fresh just before starting the hybridization steps.
31. Hybridization buffer with and without dextran sulfate and wash buffer must be discarded into a properly labeled Formamide waste container.
32. Even though there is no way to add exactly the same amount of embryos into each well, care needs to be taken to get as close as possible. If the difference in the numbers of embryos between  individual wells is too large, the wells with fewer embryos will drain more quickly than the wells with greater numbers of embryos. The embryos in the wells with fewer number will get flattened and stick together. As a result, the morphology of these will be compromised.
33. For steps 15-31 use the Q-Fill to fill the filter plates (except in steps 22&27, for these use the Electrapette 1550) and use the vacuum manifold to remove the liquid. All volumes are 200 µl  per well unless otherwise specified.
34. Set the vacuum on the lowest setting to prevent embryos from getting flattened, crushed or stuck to the membrane.
35. Seal the top of the plate with an aluminum plate sealer using the speedball roller. Then carefully seal the bottom of the plate with another aluminum plate sealer. Place the roller on the bench upside down and slide the bottom of the place on in it until a good seal is formed. This is to prevent the glycerol from leaking out of the filter plate.
36. Check individual wells to make sure that the embryos on the plate as a whole are of high quality--that the controls worked properly, that the hybridization signal quality is high (no or minimal background) and that the embryo morphology is good (e.g. embryos are not broken, flattened nor squashed).

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