Warning: Be sure both UV lamps are off before opening the gel tray or camera enclosure door (retinal hazard).
Open the camera enclosure and position the gel in the center of the
light tray, close the camera enclosure and turn on the transmitted
ultraviolet illumination (TRANS UV).
Acquire a good image of your gel under UV illumination by following the step indicated
around the Gel Doc XR interface.
Step I These controls are used for used for controling the
camera. They should be set correctly and rarely need adjustment.
Step II Set the 'Image Mode' to UV.
Step III Select 'Auto Exposure.' The camera will begin
from a very short exposure time and slowly increase the exposure until it finds
a reasonable exposure; the image of your gel will slowly emerge from darkness
as this happens. As the camera finds the optimal exposure, the exposure time
will stop increasing (watch the black bar in the lower right corner for
exposure progress). When the camera reaches optimal exposure, you can click
the 'Freeze' button to stop the camera. For most gels, the software will find
the correct exposure. If you're not sure about the exposure, you can click the
'Manual Acquire' and adjust the exposure manually.
Step IV These tools allow for the adjustment of the
image and are optional. For densitometry, it is imperative that your bands
are not saturated. Click the 'Highlight Saturated Pixels' to have the
software identify saturated pixels in red. It is fine if there is some red
around the edges of the gel or in the well, but you'll need to avoid
saturation in your bands. When you are satisfied with your image, be sure to
uncheck 'Highlight Saturated Pixels' before proceeding with the analysis.
Step V When you are finished adjusting your gel image,
click 'Analyze' to send the image from the Gel Doc XR to the analysis software.
When you see your gel image in the analysis software, you can safely turn off
the UV lamp (it will turn off by itself after a peroid of inactivity).
[Show Image]Poor Image Density ---
Saturated Bands
From the 'Lanes' menu, select 'Frame Lanes' (the keyboard shortcut is F11). Enter the
number of lanes on your gel (there are 12 lanes on the Invitrogen E-gels) and click
'Ok.'
From the 'Band' menu, select 'Create Band' (the keyboard shortcut is F9) and click on
the image of your gel at the intersection of the band of interest and the lane marker.
Alternately, you can position your cursor over a band and use the keyboard shortcut
(F9), particularly if you are identifying a large number of bands. The analysis
software will add a red horizontal at the band.
From the 'Band' menu, select 'Band Attributes...' (the keyboard shortcut is F6) and
select 'Peak Density.' The peak density will be add to the right end of the band
marker. This value is the highest intensity in the band in arbitrary units. This is
the value that you will record as the density of each band.
From the 'Band' menu, select 'Remove Band' (the keyboard shortcut is F10) and
click on the image of your gel at the intersection of the band of interest and
the lane marker. Alternately, you can position your cursor over a band and
use the keyboard shortcut (F10), particularly if you are identifying a large
number of bands. The analysis software will remove the band.
Repeat steps seven and nine for each band of interest.
Warning: Be sure both UV lamps are off before opening the
gel tray or camera enclosure door (retinal hazard).
Close the Quantity
One software and log off the computer. Turn off the UV lamp(s) and remove
your gel.
Created : 2011-06-08 12:02:00 | Last Updated: 2011-06-08 12:02:00 | Revision: 0 | 276 views