Bio-Rad PDQuest 2-D Analysis Software - Manual (Page 45)

The "Stop" button will cancel the run after the cutting tip deposits the cut in progress in its
respective microtiter plate well. The screen will give an opportunity to confirm cancel before actually
canceling the run.
5.4 Start Spot Excision
5.4.1 Begin/Resume and Pause
Press the "Begin" icon to start cutting the selected spots on the gel or membrane.
Clicking the Pause button will pause the cut run and give you the option of stopping the cut run
completely or resuming the run. After clicking on "Pause", the spot cutter will continue the cut it is
working on through eject in the microtiter plate and wash wells (if selected) before stopping. The
Pause button will be used for the "Confirm cuts" operation, described in Section 5.5.
Note: If the spot pickup rate is less than 95% of spots picked up first time, try adding more
water to the gel. Also, adjust the cutting tip height. If the tip seems to slow down and dig into the
Gel Cutting Sheet while cutting, then raise the tip height and repeat gel cuts. If it is not going
down far enough, then lower the cutting tip height.
5.4.2 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. For visible stained spots, the missed pick ups
are detected by the user looking in the microtiter plate for empty wells. If the cutting tip has not
picked up a spot, note the empty well numbers of the microtiter plate. Click on the "Recuts"
button below the image window. In the pop-up box, enter the numbers of your recut requests in
the format of: (microplate number, well number, separated by semicolons). Click on OK to close
the recut request dialog, then click on Begin to perform the recuts.
Another way to select a re-cut is to find the missed well number cut request on the screen, then
right click the mouse to prompt for re-cut.
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:
1. When UV-Fluorescent imaging has been used on the spot cutter, at the end of the cut run
the software will prompt with, "Confirm cuts?"
42