Workshop
Protocols

Soybean
EST Project

Set 384 well plates in baggie in fridge so they'll be cold while pipetting (to reduce evaporation).

Spin down samples in 96-well plates (4000 rpm, 1 min)

Turn on Hydra:

• one switch on ISO Bar power strip
• one of machine for pumps
• one on syringe machine

Hit EMPTY button on syringe unit to remove water from syringes (stored with water in syringes).

Remove white HYDRA trough that has ridges in bottom Hit pump toggle switches unit(Fill 1, Fill 2, Empty) on wash pump to remove air from the lines.

• Start with EMPTY switch on pump unit (NOT syringe unit) to empty reservoir.
• Hit FILL 1 switch on pump unit (NOT syringe unit) until no air in line
• Empty reservoir again using EMPTY on pump unit
• Hit FILL 2 switch on pump unit (NOT syringe unit) until no air in line
• Empty reservoir again.

Verify parameters for File 14:

Transferring DNA using hydra, cont.

1. Hit WASH button (upper left panel) to clean syringes.
   
   
2. Remove sample from well A1 using 10ul pipette, set aside.
   
   
3. Hit ASPIRATE/DISPENCE button (lower right panel) to form 5 ul air cushion.
   
   
4. Put 96-well plate on HYDRA stage.
   
   
5. Hit ASPIRATE/DISPENCE button (lower right panel) to aspirate 5 ul DNA.
   
   
6. Remove 96-well plate and replace it with 384-well plate that is on the special HYDRA 384-well stage adaptor. Be sure dial on stage is set to A1 quadrant Hit EMPTY button (upper left panel).
   
   
7. Repeat steps 3-7 at least once more until all solution is transferred (might need to flex 96-well plate to push up the middle-it tends to sag) for maximum of 4 transfers.
   
   
8. Add the A1 sample that was removed from 96-well plate to it's corresponding well in the 384-well plate (if 96 well plate was the A1 plate, then it goes to well A1 of 384. If plate B1, the A1 well corresponds to B1 of 384-well plate. Etc..). We do this because the HYDRA has hard time with this corner of the 384-well plate and tends to spit it onto the top of the plate instead of in the well.
   
   
9. Repeat steps 1-9 for plates A2, B1, and B2. BE SURE TO TURN DIAL ON 384-WELL STAGE FOR EACH NEW PLATE! DON'T FORGET TO START WITH THE WASH STEP!
   
   
10. Cover 384-well plates with foil tape. Spin down. Seal well. Store -20.

When finished, wash one more time. Then place white HYDRA trough on stage with fresh clean water. Hit FILL button (upper left panel). Syringes will fill with water. Leave machine with syringes full of water. Turn off all power switches, clean area, place 384-well stage adaptor on top of HYDRA and place cover back over machine.