9.3: Part II- Standard Curves
- Page ID
- 139163
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Standard curves (also known as calibration curves) show the relationship between two quantities. The standard curves are most often used to determine the concentration of “unknown” samples by comparing them to reference samples with “known” concentrations. Later in the course, we will use standard curves to measure amounts of extracted protein and to determine the size of DNA molecules. In today’s lab, you made three serial dilutions and should be able to calculate the concentrations for each dilution. Using Excel, you will prepare standard curves for each serial dilution and determine if your standard curve is accurate. Then you will determine the concentration of unknown samples, using your standard curves.
Figure 18.3. Standard Curve of Five Fold Serial Dilution
The R-squared value (R2) is the correlation coefficient or the square of the correlation. For the standard curve, this value measures how strong the linear relationship is between the reagent concentration (X-axis) and the absorbance value (Y-axis). If the R2 value = 1, then that shows a perfect positive relationship. Since your standard curves are generated from the serial dilutions you pipetted, the R2 values can also show how accurate your pipetting skills are.
Activity: Making a Standard Curve for Each Serial Dilution
- Enter the data into Excel.
- Select the data values with your mouse. On the Insert tab, click on the Scatter icon and select Scatter with Straight Lines and Markers from its drop-down menu to generate the standard curve.
- Be sure to add graph title and labels for X and Y axes.
- To add a trendline to the graph, right-click on the standard curve line in the chart to display a pop-up menu of plot-related actions. Choose Add Trendline from this menu.
- Select “display equation on chart” and “display R-squared value on chart”. Ideally, the R2 value should be greater than 0.99.
- Print the standard curves and add to your notebook.
Activity: Determining the Concentration of “Unknown” Samples
- Your instructor will have several unknown samples.
- Determine the absorbance values of each sample.
- On your standard curve, use the graph equation to solve for the corresponding concentration of these samples. Or estimate from the line graph.
| Unknown Sample | Absorbance | Concentration Compared to Serial Dilution Graphs | ||
|---|---|---|---|---|
| 2-fold | 4-fold | 5-fold | ||