The goal of this project was to reduce the agar and seed plating bottleneck from our high-throughput chemical assay. Thus allowing us to screen a higher number of compounds per hour and to allocate more time and resources to the critical phenotype screen. Using the Biomek Liquid Handling Robot we have created a protocol that effectively achieves these goals: dispensing agar and seedlings to a relatively high degree of accuracy, significantly reducing the preparation time for our phenotype screen. The general layout and procedure follows:Instrument Setup: MBP BioRobotix Widebore 220µl tips are placed in the tip loading station (TL1). A modified beaker is placed on the Hotplate to serve as the Agar dispensing station. The water in the wash station (WS1) is heated to 98.3C (see preparation). An upside-down tip box lid is placed at P14 and filled with distilled water. Empty 96-well polystyrene flat bottom plates are placed at P1-P12. A 96-well polystyrene flat bottom plate filled with 200µl seed suspension per well is placed at P13, this suspension is prepared by suspending 46.1mg of seed in 2.6ml of suspension agar per plate in duplicate. Dispensed at 12-15µl this should approximately equal 10-12 seeds per well.
Preparation: The water bath is purged by emptying the bath and refilling with distilled water. The water is then heated to and maintained at 98.3C, this heated water is routed to the wash station (WS1) and prevents clogging of the agar dispensing tips. Once the proper temperature is achieved the wash pump (WashPump1) is enabled and a user pause prompts the user to wait for thermal equilibration of the wash pump tubing. At this time agar is poured into the agar dispensing station and the heating plate is set to “8” for heating and “4” for mixing. Ensure that when aspirating at the minimum agar level the tips are above the vortex level of the agar.
Agar Dispense: New tips are loaded from the tip loader (TL1). The Biomek then aspirates 99µl of liquid agar -35mm from the top of the agar dispensing station, a tip touch is performed to remove excess agar. The Biomek then dispenses 99µl of agar into a 96-well plate. The emptied tips are then washed in water (WS1). This aspirate, dispense, wash loops continues until all the plates (P1-P12) have been filled.
Transition: A final water wash is performed (WS1) and the wash pump (WashPump1) is disabled. The tips are then unloaded (TL1) and the user is prompted to turn off the heating plate, cool the agar filled plates to room temperature and change to a new set of BMP Widebore 220µl tips (TL1).
Seed Dispense: new tips are loaded from the tip loader (TL1). The biomek then aspirates 15µl of seed suspension from P13 0.05 mm from the bottom of the well. The biomek then dispenses 15µl of the seed suspension into the 96-well plates containing 99µl agar -2.90mm from the top of the well. The seed suspension “bubble” formed during dispensing must make contact with the surface of the agar. The emptied tips are then washed at P14 through twice aspirating and dispensing 200 µl of water. This aspirate, dispense, wash loops continues until all agar filled plates (P1-P12) have been loaded with seed. The tips are then unloaded and the process is complete.
