UVP Doc-It Life Science - Manual (Page 87)

Performing 1D Analysis
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Note: Moving the bands or resizing them also changes their net intensity values. As a
result, the user will see the word "Custom" appear in the data-source column
(Concentration Calibration Window), instead of the name of the specific band.
When all lanes and bands information is deleted, the user will be asked if all
corresponding Concentration Curve data should be removed.
Performing Molecular Weight Calibration
Overview
Retardation factor Rf Lines
Applying a Molecular Weight Standard to a Lane
Removing a Molecular Weight Standard from a Lane
Overview
Molecules in an electric field migrate through a gel matrix at rates inversely proportional to the log10 of
the number of base pairs. Large molecules migrate more slowly due to large frictional force from the pore
of the matrix while small molecules migrate faster due to less frictional force.
There are many experimental conditions affecting the migration rate: gel concentration; conformation of
the DNA; applied voltage; direction of electric field; base composition and temperature; presence of
intercalating dyes; and electrophoresis buffer. It is therefore desirable to use a known molecular weight
standard as a reference to unknown samples. This marker is used to calibrate the resulting molecular
weight for each unknown bands.
Using a molecular weight (MW) marker results in a band encompassing the whole gel horizontally. This
band can be thought of as the distance traveled of a band relative to its front (Retardation factor - Rf) or
starting position. This Rf line exists for each band in the molecular weight standard. Any bands in the
unknown samples that migrate to any of these Rf lines are then compared to the Rf lines.
This section will explain how to:
Manage molecular weight standards and calibrate lanes to these standards
Determine Rf values automatically
Add Rf lines manually
Note: Rf line functions are only required if there are less than two calibrated
lanes