Bio-Rad Components for Older Model Spot Cutter - Manual (Page 24)

3.8 X, Y Calibration of Cutting Platform
The interaction between the software, the computer, the camera and the spot cutter requires
an initial calibration to bring these parts into alignment. The calibration process is designed
to be very software driven with the user loading templates and confirming to the software
that a step is ready. Then the software completes the step, and at the end the system is
calibrated and ready for use. The calibration process should be run at initial setup, if the
instrument is moved, or if there is significant changes in the accuracy over time. The
instrument has not exhibited any drift in settings, so normal use should maintain system
accuracy for over 6 months to 1 year.
To calibrate the x, y coordinates a 24 x 24 cm Coomassie stained PVDF membrane is
needed.
3.8.1 Prepare Coomassie Stained PVDF Membrane
• Cut a piece of PVDF membrane to 24 x 24 cm from a roll, Bio-Rad catalog number
162-0177. (PVDF is preferred over nitrocellulose for calibration because it is not prone
to wrinkling and shrinkage as is nitrocellulose)
• Wet the PVDF in 100% methanol in a tray, large enough for the PVDF to lay flat. Allow
the membrane to lightly shake or rock for ~2 minutes.
• Add water until it is approximately a 50% methanol solution and shake gently for
approximately 5 minutes.
• Remove the 50% methanol solution, and add undiluted Coomassie R-250 solution,
Bio-Rad catalog number161-0436, to cover the membrane. Continue gentle shaking and
turn the membrane over a few times. Allow the membrane to stain to a uniform medium
to dark blue color.
• Remove the Coomassie solution and wash with water until excess Coomassie stain is
removed. (Do not use destain solution).
• Pour off the wash water and add fresh water to keep the membrane completely wetted.
Save membrane for appropriate step in the calibration procedure.
3.8.2 Calibration of X, Y Coordinates
• From the "Basic Excision Tool" dialog box in PDQuest Spot Cutter software, select the
"Calibrate Spot Cutter" icon.
• Follow the onscreen directions for the calibration procedure.
• Before confirming the start of the calibration step using the PVDF membrane, put the
cutting mat on the platform, add a Gel Cutting Sheet and place the Coomassie stained
PVDF membrane on top of the gel cutting sheet. Make sure that all pieces are lying flat
and are completely seated inside the cutting platform (no bubbles or high spots). Place the
stainless steel Gel Holding Bars on the top and bottom of 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.
• Cover the PVDF with water by filling the cutting platform area with water to completely
cover the membrane. The water level should be a pool but not to the top of the cutting
platform edge.
• Fill a disposable Wash Tube with water and place it in the #1 well of the Wash Station in
the platform. The cutting tip will clean itself after each cut of the PVDF membrane in
this one wash tube. This is important for most efficient cutting.
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