Bio-Rad PDQuest 2-D Analysis Software User Manual
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5.3.1 Step 1: Acquire Image
There are three options for acquiring an image, "Light ON", (for visible stained gels), "Light OFF"
for PVDF blots, and "UV on (Fluor)" for fluorescent stains such as SYPRO Ruby fluorescent gel
stain
a. Acquiring an image with "Light ON", (for visible stained gels)
Use the following checklist for operation in White Light Mode:
1.
Toggle the black switch on the back of the ballast box on the spot cutter to the white
light position. See Appendix C for location of the toggle switch.
2.
Check the white lights by pushing the green "Lights on" button on the lower front
right side of the spot cutter base. If the lights do not come on under the platform,
toggle the back switch to the correct position and re-check the lights.
3.
Set the aperture to f-stop 11-16.
4.
Remove everything (including the cutting mat) from the cutting platform.
5.
Close the Enclosure door if it will be closed during imaging of the actual gel.
6.
Open the "Focus cutter camera" icon and click on the "Auto-expose" button to
automatically adjust the exposure setting. Click "OK" on the message "Auto-expose
complete" and then click "Stop".
8.
Select the "Light on (Gel)" setting in either the Basic Excision Tool or the Integrated
Excision Tool screen.
9.
Click on "Acquire Image".
Once the settings are in place, repeated gel images in the White Light mode are simply acquired
using the Acquire Image button.
b. Acquiring an image with "Light OFF" for PVDF blots
Select the Light Off to image a PVDF membrane. The platform light should be off for
PVDF because the ambient light will produce a better image than the trans-illumination of
the White Lights. Click on Acquire Image. The camera will take a single image of the gel or
membrane on the platform, and the image will appear in the window.
c. "UV on (Fluor)" for fluorescent stains such as SYPRO Ruby fluorescent gel stain.
Typical exposure times for SYPRO Ruby stained 2D gels are from 1 to 30 seconds. Start
with short exposure times and increase the time as needed for optimum imaging.
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