Douglas Instruments                                                                             Version 1.1
Optimization - using microbatch
Step by Step Instructions for Beginners

1. Clean a Nunc HLA plate with compressed air.

2. Use a ground glass syringe or pipette to dispense 6 ml of light paraffin oil into a Nunc HLA plate. Tip the plate or use a syringe etc. to remove 4.5 ml of excess paraffin, leaving paraffin in the wells. (This eliminates the possibility of droplets landing outside the wells.)

3. Place the Nunc HLA plate in the left side of the stainless steel frame on the Plate Loader as shown below.

4. Connect a 5-channel Microtip to the 5 channels of IMPAX. Place the tip in the 5-channel "collet" (holder) on the Z-arm of the Plate Loader. Place a small glass vial under the tip to collect waste.

5. Fill the ground glass syringes of the upper valves with degassed pure water and replace them.

Creating an Experiment File

6. Switch on the computer and the MCC control unit.   Click on the Start button, Programs, Douglas Instruments, then XSTEP Optimization.

7. If you want to create a new file, select File, then New Project.  Now select File again, then Save As  and provide a name for the new file.

Alternatively, to work with an existing file, select Files,   Open Project.

8. If you have created a new file, or if you want to modify the names or concentrations of the stock solutions of an old file, double click in the ingredients shown on the left of the spreadsheet.  Now modify the concentrations and names of the ingredients displayed. Fill in the viscosities of PEG solutions etc. As a guide, the viscosity of 40% PEG 8K is 40, while the viscosity of water is 1.

9. The simplest type of dispensing is when the "Laying On" option is disabled.  Select Experiment, Dispensing Parameters.  If Laying On is checked, click on the box to disable it.  (If you do use Laying On, you must use the Laying On tool to remove excess oil - see Laying On in the Protein Crystallization manual.  Laying On is more accurate.) If viscous solutions such as PEG are to be dispensed, set the Pause to 4 seconds.

10. Return to the spreadsheet. Each group of numbers shows the concentrations of the 4 ingredients in a well (other than water). Double click on a well to edit its values. To interpolate values between two wells highlight the appropriate block and select Tools, Interpolate. To optimize around a well, click on it and select Tools, Autodesign.   Wells marked for execution are colored (by default) blue.  When the experiment is ready, click on the save button to save it to disk.

Liquid Handling Calibration

11. If you suspect that any motor positions of the syringe drivers or the Plate Loader are incorrect, select Execute, Zero Motors and follow instructions.  You will now interact with a separate program for controlling the robotics called Front Panel

12. If you suspect that the 5-channel Microtip is not set to the correct height select Execute, Install Tip.  The arm(s) will move to its lowest position. Follow the instructions on the screen: (1) adjust the height of the Microtip until it is just touching the table. (2) Tighten the collet. (3) Mark the height by moving the two o-rings down to the top of the collet.

13. At the beginning of each day the system will require debubbling. Place a small bottle or vial under the Microtip in the Z-arm and select Execute, Debbuble, and follow instructions.  After debubbling syringes for about ten seconds click on the red stop button or press enter. Any air bubbles that were present at the top of the motorized syringes should have passed out into the connecting tubing.

14. Now remove the air bubbles from the connecting tubing as follows:
    a.  Remove the PTFE tubing from the needles of debubbled motorized syringes.
    b.  Expel water and air bubbles from the tubing using the ground glass syringe.
    c.  Reconnect the tubing carefully, ensuring no air bubbles re-enter.

Occasionally there may be bubbles between the upper and lower valves.  Turn the top valves to the flush position (|- ) and flush the bubbles out through the microtip with the ground glass syringes.

15. Now select Execute, Prepare syringes and follow instructions. Motorized syringes 2 - 5 will be moved to their lowest positions ready to dispense buffers etc. Motorized syringe 1 will be nearly emptied so that it is ready to suck up protein solution.

16. You now be given instructions to manually flush the bores of the microtip with solutions. The microtip acts as a reservoir of solution.  Using disposable syringes, flush all bores with water. Leave water in channels 1 and 5.   Flush the other channels with air, then flush the microtip tubing with the appropriate ingredient.

17. You must now load protein - select Execute, Load protein.  Air (or inert gas) is used to separate the protein from the water already in the tubing.  Ensure that there is no droplet on the end of the Microtip before loading air.  After loading protein, replace the Microtip in the Z-arm of the Plate Loader and tighten the thumbscrew.

Execution of the Experiment

18. Select Execute, Execute Experiment execute the experiment automatically. This will take about 5 minutes for 24 wells when viscous solutions are used.  On completion, you may wish to print the experiment - select File, Print.

Alternatively you can select a well or block of wells and right click on them to Execute selected wells.

19. After dispensing, add 5 ml paraffin to the HLA plate (or 50:50 silicone and paraffin mixture to allow slow evaporation). Place the plate in an incubator at the desired temperature.

20. Quit Front Panel before turning off your computer so that the motor positions are stored for the next use of the equipment.