24. Rinse 2x with PBT.
25. Incubate in PBT at RT with shaking 9 X for 10 min each.
26. Rinse 2x with AP buffer.
27. Wash in AP buffer at RT for 5 min; remove AP buffer.
28. Add developing solution (section 2.2.3.27)
29. Incubate with shaking until desired color development is achieved (about 75 min); remove developing solution by vacuum aspiration.
30. Rinse 3x in PBT to stop the color reaction.
31. Rinse 6x in ethanol.
32. Rinse in PBT.
33. Add 70% glycerol.
34. Store at 4 ° C 35.
35. Check individual wells on the plate under a low power magnification microscope36.
36. Embryos are ready to be photographed.
3. Notes
1. Add agar into the water and bring to boil while mixing continuously. Wait for agar to go into solution. Add base before adding grape juice. Add remaining ingredients. Mix thoroughly. Use a beaker with handles to pour hot fly food onto the prepared trays. Let the food cool to room temperature before storing at 4 ° C.
2. For each step prepare enough reaction mix for 110 reactions per plate.
3. Hydrate G-50 plate while running the PCR to save time.
4. Individual wells are very full and if the plate is not properly sealed contamination can occur. It is best to use 8-strip PCR caps rather than aluminum plate sealers to prevent cross-contamination.
5. Resuspension buffer waste must be discarded into properly labeled Formamide waste container.
6. The nylon membranes must not overlay each other to ensure proper contact with the antibody solution. Therefore, if using more than one nylon membrane per tube, the length of individual membrane sheets should be no more that 11 cm.
7. Preparation of Controls. Spot 1 µl of each of 5 dilutions (1:3.3, 1:10, 1:33, 1:100, and 1:330) of several different probes onto DIG Quantification Teststrips. Take the loaded DIG Quantification Teststrips and a DIG Control Teststrip through the quantification procedure. Evaluate which of the probe dilution series best approaches the control teststrip. Prepare an adequate amount of this probe dilution series and store at -80℃ for future use.
8. Cover the Hybaid oven door with aluminum foil. Keep the developed nylon membranes in the dark even after stopping the color reaction, otherwise they will turn completely blue over time.
9. Developing solution must be discarded into a properly labeled NBT/BCIP waste container.
10. If the probe spot is at least as intense as the 1:330 control spot the RNA probe reaction was successful and the RNA probe is ready to be used in hybridization.
11. Flies lay better in the dark. Keep the fly cages in a room without windows or cover cages with a light-tight dark cloth.
12. For maximum egg deposition make sure the fly food trays and yeast paste are at room temperature before placing in the fly cage.
13. The paste has to be easily spreadable, so that the fly food will not separate from the tray, but not too watery so that the flies will not stick to the paste.
14. Hybridization assays are conducted usingstaged embryos from 0 to 18 hrs post fertilization. Due to uneven egg deposition during this period, embryos are collected in 3 hr intervals (0-3, 3-6, 6-9, 9-12, 12-15, 15-18) to ensure equal representation of all developmental stages in the master mix made by combining equal amounts of embryos from each 3 hr interval.
15. Example of Embryo Collection Schedule
|
|
Cage #1* |
Cage#2** | |
|
|
Week 1&2 |
Week 1 |
Week 2 |
|
time |
Interval |
Interval |
Interval |
|
7:30am-10:30am |
9-12 |
3-6 |
9-12 |
|
10:30am-1:30pm |
6-9 |
0-3 |
6-9 |
|
1:30pm-4:30pm |
3-6 |
15-18 |
15-18 |
|
4:30pm-7:30pm |
0-3 |
12-15 | |
