Bio-Rad PDQuest 2-D Analysis Software - Manual (Page 31)

• Pour off the wash water and add fresh water to keep the membrane completely wetted.
Save membrane wet for appropriate step 3 in the calibration procedure.
3.3 Calibration of X, Y Coordinates
You are now ready to calibrate the spot cutter. Use the following checklist to ensure that all the
settings have been completed before beginning the calibration.
1. The lens bandpass filter is in place.
2. Camera focus has been completed. See Section 2.10.
3. The light toggle switch is set to the white light setting. Check the white lights by pushing
the green "Lights on" button on the lower front right side of the spot cutter base. If the
lights do not come on under the platform, toggle the light toggle switch to the other
position and re-check the lights.
4. The aperture setting is at f-stop 11-16 for the white light setting.
5. The cutting platform is clear of all materials.
6. "Auto-expose" has been set for the white light image acquisition setting. If not set, see
step 3.1.
7. Microtiter plate and wash well calibration has been completed. See Sections 2.13 and
2.14.
8. Cutting tip height has been tested. See Section 2.12.
To begin, Click on the "Calibrate Cutter" icon. A message will come up on the screen asking
whether the calibration is to be performed with the lens filter installed. Click "Lens filter installed" if
it is installed. This must be confirmed so that the fluorescent parts of the software will become
active. The bandpass filter must be in place before calibrating to ensure system accuracy.
• Step 1.
Remove everything from the cutting platform and make sure that it is clean and dry. This
step sets the uniformity corrections for the lighting.
• Step 2.
Place the Cutting Mat on the platform, then add the Calibration Template (clear plastic
sheet with a grid pattern). Make sure that the cutting mat and the template are clean and
dry before placing them on the cutting platform.
Note: If calibration fails at this step, it is usually due to stray marks on the Calibration template,
or an incorrect aperature/light setting.
a. Check the template for any dirt, marks, scrapes, cuts, etc. These can show up as lines in
the image and will cause the software to fail. If the template is damaged, order Bio-Rad
part number 165-7059.
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