2.1.3. PCR Product Purification (G-50 column chromatography)
1. Use MultiScreen column loader to measure out the proper amount of G-50 powder.
2. Fit a filter plate snugly on top of the MultiScreen column loader and turn upside down to transfer the powder.
3. Hydrate your G-50 plate with 300 µl of DEPC treated H20 per well and let sit at least 3 hrs, but not more than 24 hrs.3
4. 25 µl of PCR product per well plate are ready for transfer.
5. To prepare the G-50 filtration plate, place the already hydrated G-50 plate on top of a 96-well plate with alignment frame fitted on top of it. Centrifuge plates at 910 rpm for 5 min. (This will remove excess water from the G-50 plate).
6. Transfer PCR product into the prepared G-50 filtration plate.
7. Place the G50 filtration plate on top of the labeled 96-well semi-skirt PCR plate.
8. Centrifuge the G50 filtration plate with your product at 910 rpm for 5 min.
9. The samples are now ready for probe preparation
2.1.4. RNA Probe Preparation
1. Put 5 µl of 2x polymerase mix into each well of 96-well plate using a multichannel pipette.
2. Add 5 µl of PCR product using a multichannel pipette.
3. Incubate in a PCR machine at 37 ℃ for 2 hrs.
4. Add 10 µl of DNase I mix into each well using a multichannel pipette.
5. Incubate in a PCR machine at 37 ℃ for 15 min.
6. Add 20 µl of 0.2M Na2CO3 pH 10.2 to each well using a multichannel pipette.
7. Incubate at 60 ℃ in a PCR machine for 15 min.
8. Place plate on ice and quickly add 20 µl of 7.5M NH4OAc into each well using a multichannel pipette.
9. Add 160 µl of ethanol, seal well4, vortex to mix and centrifuge at 4000 rpm for one minute.
10. Incubate at room temperature for 10 min.
11. Centrifuge at 4000 rpm for 30 min at room temperature.
12. Drain by inverting plate 6X, centrifuge at 800 rpm with plate upside down to drain remaining liquid.
13. Quickly resuspend in 50 µl of resuspension buffer5.
14. Vortex at maximum speed for 25 sec and centrifuge at 4000 rpm for one minute.
15. Store at -80 ℃).
16. The RNA probe is now ready to be quantified.
2.1.5 RNA Probe Quantification
1. Using multichannel pipette spot 1 µl of resuspended RNA probe onto a positively charged nylon membrane6.
2. Using a multichannel pipette spot 1 µl of controls7 onto the same nylon membrane.
3. Crosslink in UV Stratalinker (use the autocrosslink function for steps 3 -10 use hybaid hybridization oven and tube).
4. Wash 2X with blocking solution for 5 minutes.
5. Incubate in blocking solution at room temperature for 30 minutes.
6. Incubate in a solution of 1:2000 dilution of Anti-Digoxigenin-AP Fab Fragment in blocking solution at room temperature for 30 minutes.
7. Wash 4X with blocking solution for 15 min.
8. Wash 2X with AP buffer for 5 min.
9. Develop color in the dark8 with developing solution9 (about 20 min).
10. Wash 3X in blocking solution to stop the color reaction for 3 min each.
11. Compare the RNA probes with controls (3, 10, 30, 100 and 300 pg) and determine the success rate10.
12. RNA probe is now ready to be used in hybridization.
2.2. 96-well Plate RNA in situ Hybridization
2.2.1. Embryo Collection11
1. Place a fresh tray12 of fly food covered with thin layer of yeast paste13 into a fly cage.
2. After 3 hrs. 14,15 replace the food tray with a fresh one taking care not to release any flies from the cage.
3. Store food tray with deposited embryos at room temperature for an appropriate amount of time before fixing the embryos16.
