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

Hardware Preparation

  1. Use a ground glass syringe or pipette to dispense 6 ml of light paraffin oil into a Nunc HLA plate. Tip the plate and remove 4.5 ml of excess paraffin, leaving paraffin in the wells.

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

optsetup.gif (10064 bytes)

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

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

Creating an Experiment File

  1. Switch on the computer, and wait until XTGOLD is loaded. Move to the directory where you want to keep your optimization files (e.g. C:\EXPTS\X-FILES) and press F9. Select "XSTEP - Optimization" from the menu. (Alternatively you can enter XSTEP at the DOS prompt.)

  2. The Main Menu of XSTEP will appear. If you want to create a new file, select Files, then New File and provide a name for the new file (8 letters or numbers, with no extension.)

Alternatively, to work with an existing file, select Files, then Load a File. Type in the directory that you want (if it does not appear) and press Enter. Move the cursor to the file that you want, press Enter to select it, and then Enter to load it.

Finally, press Esc to return to the previous menu.

  1. 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, press I to select the Ingredients option. Press A (or S or D for a pH experiment), then 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. Finally, press Esc, Y (to keep the changes) and Esc to return to the Main Menu.

  2. The simplest type of dispensing is when the "Laying On" option is disabled. Select Dispensing Parameters. If Laying On is enabled, press L. Press Esc to return to the Main Menu. (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.)

  3. From the main menu, select Spreadsheet. Each group of numbers shows the concentrations of the 5 ingredients in a well. Move to a well and press Enter to edit its values. To interpolate values between the two most recently edited wells select Interpolate. To optimize around a point, select Expand. See "Spreadsheet" in the manual for other options. When the experiment is ready, select Save to save it to disk. Finally press Esc to return to the Main Menu

Liquid Handling Calibration

  1. Move the cursor up to the flashing option entitled "CALIBRATION NECESSARY", and press Enter. Turn the valves to the positions shown, and press R.

  2. The positions of the motorized syringes are now shown. If you suspect that any motor positions are not correct, press Z, then S to rezero the syringes, and P to rezero the Plate Loader. Finally press Esc.

  3. 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 press D. Press Enter to start debubbling. After about five seconds press Enter again. Any air bubbles that were present at the top of the motorized syringes should have passed out into the connecting tubing.

  4. Now remove the air bubbles from the connecting tubing as follows:
    - Remove the PTFE 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.
    When this is completed, press P.

If there are 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. Then return the top valves to the fill position ( l ).

  1. A routine for preparing the motorized syringes appears. Motorized syringes 2 - 5 should be moved to their lowest positions to be ready to dispense buffers, etc. Motorized syringe 1 should be nearly empty so that it will be ready to suck up protein solution. Press P again to move syringes to these positions. When the syringes stop moving, press any key.

  2. You should now manually flush the bores of the microtip with solutions. The microtip acts as a reservoir of solution. Press Y, turn the valves to the positions shown, and press R. Using disposable syringes, flush all bores with water. Leave water in the channels 1 and 5. Flush the other channels with air, then the appropriate ingredient. Press any key, again turn the valves to the positions shown, and press R.opttip.gif (1895 bytes)

  3. You should now have returned to the Main Menu. If you suspect that the 5-channel Microtip is not set to the correct height select Liquid Handling. Press A to select Adjust/Change Microtip. The arm 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. Press Esc to return to the Main Menu.

Execution of the Experiment

  1. From the main menu, select Spreadsheet. To execute the experiment, press the Caps Lock key to obtain a new (red) menu. Ensure that there is no droplet on the end of the Microtip. Then press L to load protein, and press Enter four times to accept the default settings and load air. (The air separates the protein to be loaded from the water in the Microtip.) Undo the thumbscrew securing the Microtip, remove the Microtip from the Plate Loader, and place the tip into protein solution, avoiding any precipitate. Press any key to load the protein. Replace the Microtip in the Z-arm of the Plate Loader and tighten the thumbscrew.

  2. Press X to execute the experiment automatically. This will take about 5 minutes for 24 wells. On completion, press Y to save the file if you have not already done so. If you have a printer attached to the computer, press Y again to get a report print out. Release Caps Lock.

  3. Add 5 ml paraffin to the HLA plate (or 5 ml 50:50 silicone and paraffin mixture to allow slow evaporation). Place the plate in an incubator at the desired temperature.

  4. You must not turn off your computer without quitting XSTEP or the motor positions will be lost. To quit XSTEP press Esc until you reach the main menu. Press Esc again, then Q.

Note: The valve positions shown here indicate the desired connections between the three valve ports. For the old-style valves with levers, the lever should point to the blocked port. For example, the flush position ( |- ) corresponds to the lever pointing to the left ( ==o ) on an old-style valve.