Bio-Rad Components for Older Model Spot Cutter - Manual (Page 16)

4.3 Cutting Spots From Fluorescent Stained Gels in Basic Excision Tool
1. Check that the light toggle switch is in UV lights position.
2. Check that the aperture is open, approximately f-stop 1.4.
3. Place the fluorescent stained gel or membrane blot onto a Gel Sheet (catalog number
165-7018) and place both on the Cutting Mat on the platform of the spot cutter.
4. Place the Gel holding bars on the edges of the gel.
5. Add a thin layer of water to the gel surface.
6. Close the door.
7. Select the "UV on (Fluor)" option in the "Acquire Image" section of the Basic Excision Tool
screen.
8. Enter an exposure time, in seconds
9. Click "Acquire Image". The screen will show a processing box during image acquisition.
The amber light on the side of the Enclosure labeled, "UV lights in operation" will light
during UV illumination. There is a 15 second UV lights warm up step, then the image is
acquired, and then a dark image is acquired.
10. When the image has been acquired, it will fill in the image area of the screen. The image is
automatically inverted by the software to show the gel as dark spots with light background.
If the original fluorescent image with light spots and dark background is desired, use the
transform button to "invert image".
11. If the image does not detect enough spots or is too faint, use the transform function, or
increase the exposure time and re-image the gel or membrane.
12. The blue box shown on the image is the functional cutting area of the spot cutter. Spots
cannot be cut outside this area on the platform. If a spot is selected outside the cutting
area, an error message will occur. The cut mark will be assigned and should be moved
into the cutting region. If spots are outside the cutting area, reposition the gel and acquire
a new image.
4.4 Re-cuts in Basic Excision Tool
The specification for the ProteomeWorks Spot Cutter spot pick-up is greater than 95% effective
at picking up a spot that has been cut from a gel. There may be some spots that have not been
picked up and these can be re-cut. The efficiency of pick up on the second cut is again >95%,
so the spot is reliably picked up the second time. The missed pick ups are detected by the user
looking in the microtiter plate for empty wells. However, with fluorescent gels, the clear gel spot
(with no visible stain) is very difficult to see in the microtiter plate wells. In order to assess the
wells of the microtiter plate, the re-cut message on the screen for the fluorescent light source will
differ from the white light message. Use the following procedure for re-cutting spots in fluorescent
mode:
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