Bio-Rad PDQuest 2-D Analysis Software - Manual (Page 32)

b. Check the image quality by acquiring an image with the Basic Excision Tool screen image
acquisition. Evaluate the resulting image for light intensity. Look at the corners for any dark
shadowing. If there are shadows in the corners of the image, the bandpass filter on the
lens may be incorrectly installed.
c. Look for reflections that may come from bright, direct overhead lighting. Turn off overhead
lighting or screen the lighting from the spot cutter.
• Step 3.
Remove the Calibration Template and place a Gel Cutting Sheet on top of the Cutting
Mat. Place the wet Coomassie stained PVDF membrane on top of the Gel Cutting Sheet.
Make sure that the PVDF is lying flat and is completely seated inside the cutting platform
(no bubbles or high spots). Position the stainless steel Gel Holding Bars against the front
and back edges of the cutting platform well on the PVDF membrane. The bars should be
pushed against the platform edge. Misplacement of the Gel Holding Bars will interfere with
the cutting tip during calibration and can lead to damage to the cutting tip.
• Do not add any water to the PVDF membrane.
• Fill a disposable Wash Tube with water and place it in the #1 well of the Wash Station in
the platform. The cutting tip ejects each cut of the PVDF membrane in this one wash
tube. This is important for most efficient cutting.
• Press the onscreen start key to begin the calibration process.
• The Spot Cutter will proceed to cut 25 spots in an array of 5 rows with 5 spots per row.
• Once all 25 spots are cut, the system will automatically acquire an image and calibrate
itself.
• A dialog box will appear stating that the calibration is successfully completed. Press OK,
and the process is complete. You are now ready to begin Spot Cutting.
Note: If Step 3 fails
Calibration will fail if any of the 25 spots are not cut. Uncut spots are usually due to the cutting tip
not cutting deeply enough. If many of the holes did not have score marks or were barely scored,
adjust the cutting tip depth to slightly down from the present position. See Section 2.12 for cutting
tip height adjustment. Once the cutting tip height has been adjusted, restart the calibration procedure.
The calibration will remain in place for many months of routine spot cutting. However, if there is
excessive vibration on the lab bench from instruments other than the spot cutter, or if the spot
cutter and/or camera is moved or bumped, evaluate the accuracy of the cutting. If it is unacceptable,
repeat the calibration procedure. Periodically evaluate the accuracy of cutting with a gel or a piece
of paper with spots drawn on it. Target the spots and evaluate the performance. If the spot cutter
is off target, recalibrate the instrument.
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