Bio-Rad EXQuest Spot Cutter - Manual (Page 37)

Figure 5-2. Manual excision tool.
7. Close the spot cutter front door and click Acquire Image to take an image of the gel tray contents.
Do not open the door during image acquisition. As a safety precaution, the UV EPI lights will
automatically turn off if the door is opened. Images acquired with "UV EPI" are automatically
inverted by the software to display dark spots with a light background. If the image does not detect
enough spots or is too faint, use the transform function to optimize the image contrast (see Section
8.3).
8. Identify the cutting tip size, 1.0 mm or 1.5 mm, in the "Cut Options" panel (see Section 8.4). Note
that the cut request circle size displayed on the image depends on the tip size selected.
9. From the "Cut Options" panel, specify the type of gel to cut (see Section 8.4).
• Free Gels. Select this option to use a protocol optimized for cutting non-backed gels.
• Plastic-backed Gels. Select this option to use a protocol optimized for cutting plastic-backed
gels. When this option is used, gel plugs are cut and then dislodged from the gel backing by a
side-to-side cutting tip motion.
• Glass-backed gels. Select this option to use a protocol optimized for cutting glass-backed gels.
When this option is used, gel plugs are cut and then dislodged from the gel backing by a
side-to-side cutting tip motion. Note that the cutting tip is raised by 3 mm to accommodate the
glass plate.
• PVDF: This option is available only when the spot cutter is in nitrocellulose membrane cutting
mode, and is used to specify that either a PVDF or Nitrocellulose membrane will be cut. When
cutting blots:
– Fill microplate wells with DI water to within 1 to 2 mm of the top. In order to release the cut
membrane into a microplate well, the cutting tip must be in contact with water.
– Do not overfill the wells/tubes as this will allow cut membrane to float between adjacent
wells.
10. Optional. Select Make multiple cuts on large objects in the "Cut Options" panel (see Section 8.4)
to harvest large amounts of protein from a single spot and deposit it into a single microplate well.
Enter the maximum number of cuts that you want to allow in the Maximum cuts/object field.
11. Select the microplate format to be used from the "Plate Options" panel (see Section 8.5), and then
specify the well volume and well loading order. The well volume will determine the maximum number
of spots that can be placed in a single well/tube.
MANUAL EXCISION TOOL
5-2