Imaging and Quantifying Gels

Staining

  • Inspect stain and destain solutions to make sure that they are fresh and free of broken gel pieces
  • To make new stain :
    • Pour out existing stain into ethidium bromide waste container
    • Rinse plastic with MilliQ water, pouring rinse into waste container
    • Fill plastic with approximately one liter of MilliQ, and add a few drops of ethidium bromide until water is slightly colored
  • To make new destain:
    • Pour out existing destain into ethidium waste container
    • Rinse plastic with MilliQ water, pouring rinse into waste container
    • Fill plastic with appoximately one liter of MilliQ water
  • Soak the gel in ethidium bromide stain for 30 minutes
  • Use spatula to move gel into a Milli-Q water destain, and let sit for 15 minutes

Positioning Gel

  • Turn on FluorS-MultiImager (power switch is located on back-right hand side)
  • Thoroughly wet the imager’s glass plate with water
  • Remove gel from destain, rinse with Milli-Q water to remove any loose pieces of agarose, and move onto the wet surface of the imager
  • Change gloves
  • Wearing clean gloves, sit at computer and click on “Quantity One” icon (a red icon, found most often on the upper right side of the Desktop)
  • Select “fluorese” from “File” menu
  • Click on “Position” button
  • Use the red plus-mark and the blue grid to center and align the gel
  • Click on “Stop” button and close door to FuorS-MultiImager

Creating Image

  • Set exposure time to 10.0 and click on “Preview” button
    • If bands are too faint, increase exposure time and re-preview
    • If gel is too bright, either decrease exposure time or destain longer
  • Click on “Acquire” button
  • Save image by selecting “Save” from “File” menu, and/or print image by going to “Print” (also under the “File” menu) and selecting “Print Image,” then clicking on “Go” button

Quantifying Bands

  • Open “Lane Tools” and “Band Tools” toolbars (16th and 17th icons from left, respectively, on main Quantity One toolbar)
  • Zoom in on gel (7th icon from left, on main toolbar) as much as possible without cutting off any bands
  • Mark all lanes of interest
    • Select “Create Lane” (6th icon from left, on Lane Tools)
    • Position the mouse above the bands in first lane of interest, centering it horizontally
    • Click, hold, and drag the mouse down past the end of the last band within that lane
    • There should now be a red line running through the center of each band within the first lane
    • Click on “Adjust Lane” (7th icon from left, on Lane Tools)
    • Position mouse over least centered section of line
    • Click, hold and drag the mouse to adjust the line as needed
    • Repeat with all relevant lanes
  • Click on “Detect Bands” (1st icon from left, on “Band Tools”), click on “Close” (all bands should now have brackets roughly surrounding them)
  • Click on “Remove Band” (4th icon from left, on Band Tools)
  • Click on a set of brackets that does not correspond to an actual band, click on “Yes,” and repeat for all other incorrect brackets
  • Adjust brackets vertically
    • Click on “Adjust Bands” (3rd icon from left, on “Band Tools”)
    • Click and hold on the upper bracket corresponding to the first band
    • Adjust bracket both visually and by using the pop-up graphs
    • VISUALLY: The bracket should serve as the upper/lower limits to the bands
    • GRAPHS: Each band has its own peak on the graph. The brackets should be set so that only the steepest section of each peak is highlighted in yellow.
    • NOTE: In theory, each one of these approaches should yield the same result. In practice, such is rarely the case, so it is important to rely on a combination of the two.
  • Adjust brackets horizontally
    • Click on “Lane Width” (10th icon from left, on “Lane Tools”)
    • Click on first lane
    • Arbitrarily adjust number and click “OK”
    • Continue this process until brackets serve as borders to the bands
    • If the (red line demarking the) lane is not centered over the bands it will have to be re-aligned, as it will be impossible to properly adjust the brackets otherwise.
    • NOTE: Re-aligning the lanes will erase the brackets. This time, when preparing to “Detect Bands,” change the “Lanes” option from “All” to “One”, and type in the re-adjusted lane’s number, as labeled by Quantity One.
  • Select “Quantity Standards” from “Analysis” menu, and click on “Create New”
  • Click on the button corresponding to “Select Bands”
  • Return to the window with the gel imange and click on bands from lane containing the mass standard
  • Return to the “Quantity Standards” window
  • Set “Interpolation” to “Linear Regression,” and “Extrapolation” to “Yes”
  • Manually alter the “Quantity” column of the table at the bottom of the window to reflect the known values (if four lanes appear, the values are 20, 15, 10, and 6 for each 1µL added)
  • Click on “Show Curve”
  • If any point is very off, remove it from the chart
  • Click on the button corresponding to “Calibrate Gel,” and click on “OK”
  • Select “All Lanes Report” from “Report” menu, and select “Report”
  • Print report if necessary by choosing “Print All” (2nd icon from left, at bottom of window)
  • Save calculations in personal folder by selecting “Save” from “File” menu (calculations and gel image are all saved together)
  • NOTE: Quantity One caculates the concentration (see “Calibrated Quanitity” column in the “Report” printout) in terms of ng/mL
  • Quit program

Cleanup

  • Remove gel from FuorS-MultiImager and either store in Ziplock bag or throw away in ethidium bromide waste container
  • Clean off FluorS-MultiImager with ethanol
  • Remove any broken pieces of gel from stain and destain, place in ethidium bromide waste container
  • Throw gloves away in ethidium bromide waste container