32-Well + Thermogenetics Box

This behavioral apparatus adapts the 32-well behavior chambers used for Drosophila copulation duration and courtship assays to be compatible with thermogenetic manipulations. This set-up utilizes a water reservoir that sits below the floor of the behavior chambers, allowing the experimenter to control the temperature within in the well by manipulating the source of the water. The reservior is connected to a water bath that can be set to warmer temperatures, from which water is continuously pumped in a closed loop from the bath to the reservior and back to the bath.

The 32-WellThermogeneticsBox_BehaviorChambers files contain schematics and laser cutter settings for producing the 32-well plate with water reservior.

The 32-WellThermogeneticsBox_BehaviorChambers files contain schematics and laser cutter settings for producing a platform on which we sit the 32-well plate with water reservior.

A commercially available camera can be placed above the apparatus to record behavior.

Parts List

See the excel sheet for easy copy and paste to McMaster-Carr's order form and for notes.

1x 1/6" Clear Acrylic: McMaster-Carr #8589K12

5x 1/8" Clear Acrylic: McMaster-Carr #8560K257

4x 1/8" Black Acrylic: McMaster-Carr #8505K755

1x 18-8 Stainless Steel Hex Head Screws, 4-40 Thread Size, 3/4" Long: McMaster-Carr #92314A113

1x Brass Flanged Knurled-Head Thumb Nut: McMaster-Carr #92741A100

Loctite 587 Gasket Maker Sealant: McMaster-Carr #7660A21

1x (10 ct) Plastic Barbed Tube Fitting for Air and Water, Tight-Seal, for 1/8" Tube ID x 10-32 Thread Male Pipe: McMaster-Carr #5463K53

1x 10 ft Tygon PVC Soft Plastic Tubing for Air and Water, 1/8" ID, 1/4" OD: McMaster-Carr #6516T14

1x (10 ct) Plastic Barbed Tube Fitting for Air and Water, Tight-Seal, Reducer, for 1/4" x 1/8" Tube ID: McMaster-Carr #5463K628

1x 25 ft Clear Masterkleer Soft PVC Plastic Tubing for Air and Water, 1/4" ID, 3/8" OD: McMaster-Carr #5233K56

1x (10 ct) Plastic Barbed Tube Fitting for Air and Water, Straight Reducer, for 3/8" x 1/4" Tube ID: McMaster-Carr #5372K517

1x 10 ft High-Pressure Soft PVC Plastic Tubing for Air and Water, 3/8" ID, 5/8" OD, Clear: McMaster-Carr #5238K748

1x EcoPlus Eco 185 Water Pump Fixed Flow Submersible Or Inline For Aquariums, Ponds, Fountains & Hydroponics - UL Listed, 158 GPH, Black: Amazon https://www.amazon.com/EcoPlus-Submersible-Aquarium-Fountain-Hydroponics/dp/B0018WVNXC

1x Water Bath, such as PolyScience WB05A11B Digital General Purpose Water Bath, 5 L Capacity, 120V/60 Hz: Amazon https://www.amazon.com/PolyScience-WB05A11B-Digital-General-Capacity/dp/B00N5ILOK4

You will need access to a threader, scissors, and tape. You will also need access to a tool (such as a thermocouple thermometer) that can measure the temperature in the wells.

Assembly

32-Well Plate with Water Reservior Assembly

1. Cut all the behavior chamber acyrlic pieces according to the specifications in the schematic files. You can discard the ellipsoid shaped insert from the water reservior layer, but keep the smaller donut shaped center piece.
2. Thread the two holes that were not rastered in a hexagon shape so that the 10-32 thread male connecter can be screwed into place.
3. Insert all the screws so that the hexagonal head sits in the rastered inset on the bottom. Flip the acrylic over (you may want to put something against the screw heads or tape them in place so they stay put). You should end up with the acrylic laying flush or almost flush to the top of your workspace and all the screws sticking out pointing upwards.
4. Stack the water reservior layer on the bottom, making sure all the screws go through the appropriate holes. Put the donut shaped center piece on the center screw.
5. Affix the cut well bottoms/diffuser paper layer to the water reservior layer by coating all the remaining water reservior pieces (the rectangle with the elipsoid shape removed and the small donut center piece) with liquid gasket and pressing the diffuser paper down. Smooth it out so there are no gaps.
6. Stack the well layer, then the door holder layer with the doors, then the top layer on the well bottom/diffuser paper layer.
7. Use thumb nuts on all screws to secure all the layers in place.
8. Flip the whole plate over so that the bottom is facing up. Then screw in the threaded connector (McMaster-Carr #5463K53) so that the barbed portion that will connect to tubing is pointing out. Put a little liquid gasket around the connection to seal it.
9. Let the whole thing dry for at least 30 mins.

Platform Assembly

1. Cut all the platform acyrlic pieces according to the specifications in the schematic files. You can discard the 2 small square-ish cutouts from the longest sides (but keep track of which sides are which).
2. Attach the sides to the bottom and secure with tape. The long sides should be positioned so that the jigsaw end is flush with the shorter side and the smooth end starts a gap before the next short side. You want the gap after the smooth edge to be in different places so that you can not see though the whole side like so:

3. Attach the top and secure with tape.

Connecting to a Water Source

1. Cut two 8" pieces of small tubing. Attach the each piece of tubing to one of the connector protruding from the bottom of the behavior chamber.
2. Sit the behavior chamber on top of the platform and position the tubing so that each piece of tubing comes out a different gap in the side.
3. Position the water bath next to the platform.
4. Connect the small tubing to the medium tubing with connectors - cut the length of the medium tubing so that it can reach the water bath.
5. Pick one tube to be the output, tape that one to the side of the water bath so water flowing from that tube will be deposited in the bath.
6. The other tube is the input, connect that tube to a ~4" piece of high pressure large tubing using a connector.
7. Connect the high pressure tubing to the pump, using an adaptor that came with the pump, and place the pump in the water bath.
8. Fill the water bath, and turn on the pump. You may need to temporarily raise the side of the behavior chamber plate with the output tubing to encourage the air pocket to be pushed out.
9. We use a 30 degree water bath to achieve a 28.5 degree well temperature (measured with a thermocouple pressed against the well floor) because the water will cool slightly once it leaves the bath. You will need to confirm the temperature in your well and set the bath accordingly.