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BDGP: 96-well RNA In Situ Hybridization Protocol
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96-well Hybridization Protocol

Probe Preparation

Cell Inoculation

1.     >Add 176 ml of 50 mg/ml antibiotic to 110 ml of SB and mix thoroughly

2.      Add 1.0 ml of antibiotic /SB mix into each well of Beckman block using a multichannel pipette

3.      Add 10 ml of thawed cells per well using a multichannel pipette (Brand)

4.      Seal tightly with Airpore Tape Sheets

5.      Place Beckman block in a shaker incubator for 18 hrs at 37 °C at 300 RPM

6.      Store at 4°C

7.      Cells are now ready for PCR

PCR

1.      Dilute cells 1:100 in dd H20

2.      Mix all reagents thoroughly

3.      Add 29 mL of the mix to each well using a multichannel pipette

4.      Add 1 mL 1:100 cell dilution into each well using a multichannel pipette, mix thoroughly and spin down the plate again

5.      Place plate in PCR machine

6.      Run Gel

7.      The samples are now ready for G-50 purification

 

Links to different cycling conditions:

PCR Protocol for Genomic DNA

PCR Protocol for BSK

PCR Protocol for pFLC 1 polylinker

PCR Protocol for pOT2A

 

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 mL of DEPC treated H20 per well and let sit at least 3 hrs, but not more than 24 hrs.

4.      25 mL of PCR product per  well plate are ready for transfer

5.      Prepare G-50 plate. Place already hydrated G-50 plate on top of a 96-well U-well plate with alignment frame fitted on top of it. Spin down plates for 5 min @ 91 0. (This will remove excess water from the G-50 plate).

6.      Transfer PCR product into the prepared G-50 plate

7.      Place the G50 plate on top of the labeled 96-well semi-skirt PCR plate

8.      Spin down the G50 plate with your product for 5 min @ 910 rcf

9.      The samples are now ready for probe preparation

RNA Probe Preparation

1.      Put 5 mL of 2x polymerase mix into each well of 96-well plate using a multichannel pipette

2.      Add 5 mL of PCR product using a multichannel pipette

3.      Incubate for 2 hrs. @ 37 °C in a PCR machine

4.      Add 10 mL of Dnase I mix into each well using a multichannel pipette

5.      Incubate for 15 min. @ 37 °C in a PCR machine

6.      Add 20 mL of 0.2M Na2CO3 pH 10.2 to each well using a multichannel pipette

7.      Incubate for 15 min. @ 60 °C in a PCR machine

8.      Place plate on ice and quickly add 20 mL of 7.5M NH4OAc- into each well using a multichannel pipette

9.      Add 160ul of Ethanol, seal well vortex to mix and spin down

10.  Incubate for 10 min. @ RT

11.  Spin for 30 min. @ RT @ 4K

12.  Drain by inverting plate 6X, spin up to 800 rpm with plate upside down to drain remaining liquid

13.  Quickly resuspend in 50 mL of resuspension buffer

14.  Store @-80 °C

15.  The RNA probe is now ready to be quantified

RNA Probe Quantification

1.      Using multichannel pipette spot 1 mL of resuspended RNA probe onto a positively charged nylon membrane

2.      Using a multichannel pipette spot 1mL of controls[i] onto the same nylon membrane

3.      Crosslink in UV Stratalinker (use the autocrosslink function

for steps 3 -10 use hybaid hybridization oven and tubes

4.      Wash 2X for 5 min. with blocking solution

5.      Incubate for 30 min. @ RT in blocking solution

6.      Incubate for 30 min. @ RT 1:2000 dilution of Anti-Digoxigenin-AP Fab Fragment in blocking solution

7.      Wash 4X for 15 min. with blocking solution

8.      Wash 2X for 5 min. with AP buffer

9.      Develop color in the dark with developing solution (about 20 min)

10.  Wash 3X for 3 min.  in blocking solution to stop the color reaction

11.  Compare the RNA probes with controls and determine the success rate

12.  RNA probe is now ready to be used in hybridization

Preparations of Embryos

Hybridization assays are conducted with staged embryos from 0 to 18 hrs after fertilization. Due to an 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 stages in the master mix made by combining equal amounts of embryos of each 3 hr. interval

Embryo Collection[ii]

1.      Place a fresh tray with fly food covered with thin layer of yeast paste into a fly cage

2.      After 3 hrs. replace the food trey with a fresh one taking care not to release any flies from the cage

3.      Store food tray with deposited embryos at RT for an appropriate amount of time before fixing the embryos

Embryo Fixation

1.      Keep embryos of different time periods separate during fixation

2.      Wash embryos off the food trey into a three level sieve to remove fly food and yeast paste

3.      Fill reservoir with 100% bleach and shake embryos in for 3 min. to de-chorionate

4.      Wash well with distilled water

5.      Remove embryos form the sieve using a spatula and place into a 50ml falcon tube filled with 50-50 mix of Heptane and 4%Formaldehyde/PBS

6.      Incubate 25 min. with shaking

7.      Remove lower aqueous phase and replace with equal volume of Methanol

8.      Shake for 1 min then allow embryos to settle. DO NOT VORTEX

9.      Remove upper phase along with vitelline membranes and embryos remaining at interpahse

10.  Remove remaining Methanol

11.  Wash 3x in Methanol

12.  Top off 50ml Falcon tube with Methanol

13.  Store Embryos @-20 °C

14.  Embryos are now ready to be used in hybridization

96-well Plate RNA in situ Hybridization

1.      Prepare a master embryo mix by combining equal amounts of embryos from all six time periods in a 50 ml Falcon tube

2.      Take out 1ml of embryos per plate from the master mix and place them in 15 ml Falcon tube

3.      Re-hydrate for 2 min in 3:1 Methanol:4%Formaldehyde/PBS

4.      Re-hydrate for 5 min in 1:3 Methanol:4%Formaldehyde/PBS

5.      Fix for 10 min in 4%Formaldehyde/PBS

6.      Rinse 6x in PBT

7.      Add 3 mL of Hybridization Buffer WITHOUT Dextran Sulfate per 1 mL of embryos

8.      Rock for at least1 hr @ RT to pre-hybridize embryos

9.      During pre-hybridization put 200 mL of Hybridization Buffer WITH Dextran Sulfate in a 96 U-well plate using Multichannel pipette

10.  Add 2 mL of probe to into each well in columns 1-11. Add 2 mL of control probe into wells in column 12. (Do not add control into at least 1 well in column 12--negative control )

11.  Mix thoroughly and spin down

12.  Carefully pour pre-hybridized embryos into a 25 mL reagent reservoir

13.  Add 20 mL of embryos into each well of 96-well filter plates (using a multichannel pipette with wide opening tips)

14.  Transfer the probes from the 96 U-well plate into the filter plate and seal the filter plate

15.  Rock overnight @ 55 °C

16.  Add 100 mL of warm Wash Buffer

17.  Vacuum out the hybridization buffer-wash buffer mix; once all the liquid is removed from wells quickly turn off the vacuum

18.  Rinse 2x with wash buffer

19.  Rock 8x for 30 min @ 55 °C in wash buffer

20.  Rock overnight @ 55 °C in wash buffer

21.  Rinse in PBT

22.  Rock for 30 min @RT in PBT; remove PBT

23.  Rock for 2 hrs @RT in PBT, 5% Goat Serum, 1:2000 dilution Anti-Digoxigenin-AP Fab Fragments

24.  Rinse 2x with PBT

25.  Rock/wash 9x for 10 min @RT in PBT

26.  Rinse 2x with AP buffer

27.  Wash for 5 min @RT in AP buffer; remove AP buffer

28.  Add developing Solution

29.  Incubate with rocking until desired color development is achieved (about 75 min.); remove developing solution

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 @ 4 °C

35.  Check individual wells on the plate under a low power magnification microscope

36.  Embryos are ready to be photographed



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