User`s manual

Alpha Innotech User’s Manual, version 4
5-50
Gel Smiling Correction with Rf Curve Tool
The AutoLane portion of the software contains the Rf Curve tool which allows the user to
adjust for gel smiling or curvature. Using this tool will increase the accuracy of
Molecular Weight calculations and Automatic Band Matching. After the Rf Curves have
been applied a new column of data will be generated, Adjusted Rf. The Rf. values
reported are relative values from 0 to 1 where 0 is the origin and 1 is the lane front
without correction for gel smiling as commonly occurs in gel electrophoresis. The
Adjusted Rf. (Adj. Rf.) is the corrected value.
Suggestions:
The software calculates Adjusted Rf. based on the bands position relative to the nearest
Rf Curve. When the band lies between two Rf curves with different curvatures a
weighting is applied where greater weight is given to the nearest Rf Curve. For the most
accurate calculations enough curves should be added to properly mimic the curvature of
the sample. We also suggest placing at least one Rf Curve above the top most band and
below the bottom most band. If these curves are left out the software will use the lane
origin and front as the first and last curves. These are assumed to be straight.
Applying Rf. Curves
After identifying lanes in AutoLane the following dialog box appears.
Select the Rf Curve Tool by clicking the associated button.
Click on the image to apply the curve. Clicking within the lane boundaries will produce
a three point curve with an anchor point each on the outside of the first and last lanes and
one at the position of the mouse click. Clicking outside the lane boundaries to apply a
curve will create a two point curve with anchor points on either extreme.