ORYX Microbatch Screening Ran Meged -1-

Microbatch oil protein & reagent solution -2-

Microbatch oil protein & reagent solution -3-

Screening Routine Use a 2-bore microtip -4-

Screening Routine Use a 2-bore microtip Start with both bores full of water -5-

Screening Routine Air bubbles Use a 2-bore microtip Start with both bores full of water Suck up 1µl of air into both channels -6-

Screening Routine Air bubble Protein slug Use a 2-bore microtip Start with both bores full of water Suck up 1µl of air into both channels Suck up protein required for experiment µl -7-

Microbatch screening – dispensing cycle Screening solutions Target plate -8-

Microbatch screening – dispensing cycle (1) 1.Pick up screening solution -9-

Microbatch screening – dispensing cycle (2) 1.Pick up screening solution 2.Transfer to microbatch drop -10-

Microbatch screening – dispensing cycle 1.Pick up screening solution 2.Transfer to microbatch drop 3.Add Oil Drop + oil (3) -11-

Microbatch screening – dispensing cycle 1.Pick up screening solution 2.Transfer to microbatch drop 3.Add Oil Drop 4.Rinse (4) -12-

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Hardware preparation: Clean a Douglas Vapor Batch [VB] plate with compressed air, and place it on the top left corner of the Plate Loader. A A -14-

Hardware preparation: Clean a Douglas Vapor Batch [VB] plate with compressed air, and place it on the top left corner of the Plate Loader. Place a Nunc Plate to the right of the VB plate. up to 96 screening solutions. A B A B -15-

Hardware preparation: Clean a Douglas Vapor Batch [VB] plate with compressed air, and place it on the top left corner of the Plate Loader. Place a Nunc Plate to the right of the VB plate. up to 96 screening solutions. Place the rinse block to the right of the Nunc Frame, with bottles in wells 1 to 5. Fill the first 3 bottles with clean water for rinsing. Fill the 5th bottle 75% full with Oil. (Leave the 4th bottle empty for waste(. A B C A B C -16-

Hardware preparation: Clean a Douglas Vapor Batch [VB] plate with compressed air, and place it on the top left corner of the Plate Loader. Place a Nunc Plate to the right of the VB plate. up to 96 screening solutions. Place the rinse block to the right of the Nunc Frame, with bottles in wells 1 to 5. Fill the first 3 bottles with clean water for rinsing. Fill the 5th bottle 75% full with Oil. (Leave the 4th bottle empty for waste(. Connect a 2-channel Microtip to the first two channels (green and red). Place the tip in the 2-channel "collet" (holder) on the left (Z) arm of the Plate Loader. A B C D A B C -17-

Hardware preparation: load the program Front Panel, which is used for controlling the robotics. Front Panel -18-

Hardware preparation: If you suspect that any motor positions of the syringe drivers or the Plate Loader are incorrect, select MCC (motion control center), Rezero All Motors and follow instructions. Front Panel -19-

Hardware preparation: Select Syringes for Debubble and Rezero… Front Panel -20-

Hardware preparation: Select PlateLoader for testing and installing tips, Rezero, move plate loader to home position… Front Panel -21-

Hardware preparation: Select Protein for Loading and recovering Protein… Front Panel -22-

Hardware preparation: Select Options for Execute in Background Front Panel -23-

Hardware preparation: Debubbling At the beginning of each day the system will require Debubbling. Place a small bottle or vial under the Microtip in the Z arm, select Syringes, Debubble, and follow instructions. Any air bubbles that were present at the top of the motorized syringes should have passed out into the connecting tubing. Motorized Syringe (100µl) Upper valve Lower valve Ground glass syringe (10ml) (one of the) 2-Channel microtip -24-

Hardware preparation: Debubbling follow instructions and remove the air bubbles from the connecting tubing as follows: -25-

Hardware preparation: Debubbling Remove tubing from the needles of debubbled motorized syringes. Expel water and air bubbles from the tubing using the ground glass syringe. Reconnect the tubing carefully, ensuring no air bubbles re-enter. -26-

Hardware preparation: Debubbling Occasionally there may be bubbles between the upper and lower valves. Turn the top valves to the flush position Flush the bubbles out through the microtip with the ground glass syringes. -27-

Hardware preparation: Debubbling Use Lower syringe to flush bubbles through the microtip and to fill microtip with water or solution. -28-

Software Preparation Microbatch repeat well.xpp Repeats single screen well from Nunc F8 plate to chosen block on Microbatch plate. Microbatch screening.xpp Dispenses Microbatch screen from Nunc F8 plate to Microbatch plate. Protein + solution droplets are subsequently covered with oil if "Auto oil on" is checked. Microbatch transposed.xpp Dispenses Microbatch screen from SBS Format 96 well plate to Microbatch plate. Screen 'block” is transposed to keep the numbering the same on both plates. E.g. Stock solution A3 will end up in well A3 on the destination plate. Additive screening.xpp Dispenses Microbatch screen with additives from Nunc F8 plate to Microbatch plate. Precipitant in Channel 3 (Blue) -29-

Software Preparation The program Wasprun will load If desired, change the size of the block on the Source plate and / or the location of the block on the Destination (VB) Plate. You can also change the drop volume, concentration and dispensing conditions. When your experiment is ready, Select “Dispense”. -30-

Running the Experiment The system will now check the positions of the motorized syringes. If necessary, you will be instructed to turn valves and the syringes will be moved in order to carry out the experiment. -31-

Running the Experiment A page of configuration information and instructions will now appear. Note that all air bubbles must be removed from all tubes of the first two channels including the microtip. Remember to remove any droplet on the end of the microtip to prevent this from being sucked in later on. -32-

Running the Experiment Air will now be loaded into the microtip to separate the protein from water already in the microtip. After loading air, you will be asked to provide protein. Place the protein in the position chosen on the Protein Load Plate. The tip will automatically load the protein. Wipe the tip after the loading is complete if required. Click OK to carry out the experiment automatically. -33-

Running the Experiment When it is finished, "top up" the VB plate with 4-5 ml Al’s Oils (50:50 silicone and paraffin mixture) or 100% paraffin to reduce evaporation. Place the plate in an incubator at the desired temperature. Quit Front Panel before turning off your computer so that the motor positions are stored for the next use of the equipment. Turn off the MCC by pressing the power button on the front of the machine. -34-

Maintenance Flushing Microtip after use The Microtip should be thoroughly flushed after each session. Place a 1ml (or 3ml) syringe containing distilled water in each valve in the lower row, turn the valve, and press the plunger in firmly. Repeat this at least three times. Finish by passing air through the Microtip and disconnecting. Store it coiled up and flat to avoid bending of the tip. Microtip cleaning Certain proteins may have a tendency to coat the inside of a microtip. This may cause the air bubble (that is used to separate the protein sample from the water in the microtip) to become stuck or to break up. Tips that are coated with protein can be cleaned by flushing first with 1 M NaOH, then with buffer solution to get rid of the alkali. If this procedure does not work, try conc. HCl mixed with an equal volume of methanol (again followed with buffer to remove the acid). -34-

Maintenance Flushing Microtip after use The Microtip should be thoroughly flushed after each session. Place a 1ml (or 3ml) syringe containing distilled water in each valve in the lower row, turn the valve, and press the plunger in firmly. Repeat this at least three times. Finish by passing air through the Microtip and disconnecting. Store it coiled up and flat to avoid bending of the tip. Microtip cleaning Certain proteins may have a tendency to coat the inside of a microtip. This may cause the air bubble (that is used to separate the protein sample from the water in the microtip) to become stuck or to break up. Tips that are coated with protein can be cleaned by flushing first with 1 M NaOH, then with buffer solution to get rid of the alkali. If this procedure does not work, try conc. HCl mixed with an equal volume of methanol (again followed with buffer to remove the acid). Clean Clean Clean Clean and then… clean some more. -34-