03 - Sweet Laboratory: Exploring Food Science with Biotechnology
Module III: STEM-Based Data Analysis of Food Dyes Using a Standard Curve
Agarose gel electrophoresis separates biomolecules into discrete bands, each comprising molecules of the same size. How can these results be used to deter- mine the lengths of different fragments? Remember, as the length of a biomolecule increases, the distance to which the molecule can migrate decreases because large molecules cannot pass through the channels in the gel with ease. Therefore, the mi- gration rate is inversely proportional to the length of the molecules—more specifi - cally, to the log 10 of molecule's size. To illustrate this, we ran a sample that contains bands of known lengths called a “standard”. We will measure the distance that each of these bands traveled to create a graph, known as a “standard curve”, which can then be used to extrapolate the size of unknown molecule(s). 1. Measure and Record Migration Distances Measure the distance traveled by each Standard Dye Molecule from the lower edge of the sample well to the lower end of each band. Record the distance in centime- ters (to the nearest millimeter) in your notebook. Repeat this for each dye fragment in the standard. Measure and record the migration distances of each of the fragments in the un- known samples in the same way you measured the standard bands.
A B C
Figure 2-3: Measure distance
migrated from the lower edge of the well to the lower edge of each band.
2. Generate a Standard Curve. Because migration rate is inversely proportional to the log 10 of band length, plotting the data as a semi-log plot will produce a straight line and allow us to analyze an exponential range of fragment sizes. You will notice that the vertical axis of the semi-log plot appears atypical at first; the distance between numbers shrinks as the axis progresses from 1 to 9. This is because the axis represents a loga- rithmic scale. The first cycle on the y-axis corresponds to lengths from 100-1,000 base pairs, the second cycle measures 1,000-10,000 base pairs, and so on. To create a standard curve on the semi-log paper, plot the distance each Standard Dye Molecule migrated on the x-axis (in mm) versus its size on the y-axis (in base pairs). Be sure to label the axes!
QUICK REFERENCE: The Standard dyes have the fol- lowing base pair equivalents.
Blue Red
5000 3000 1000
Purple Orange
500
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