Let's Make A Buffer

Let's Make a Buffer

Instructor: Dr. Stone
Submit report to: turnitin.com
Points: 100

The goals of this project:

1.Make each of these Buffers for subsequent experiments:
a. 50 mL of 5X and then 1.0 L of 1X TAE buffer for gel electrophoresis of DNA, use this website to learn more: TAE information
b. 1.0 L 50 mM Tris Nitrate pH 7.0 buffer, send to autoclave.
c. 10 mL of 100 mM Phosphate buffer, pH 7.4, for the Microsome assay experiment.

2. Build a spreadsheet that allows you to quickly calculate the amount of each reagent needed to make a phosphate buffer, you should be able to input volume, molarity and pH and then your spreadsheet will calculate the amount of each reagent needed.

3. Write detailed instructions for making the the 100mM phosphate buffer. Write these instructions for someone who has never taken a college chemistry class. Explain everything.

4. How would you test the buffering capacity of your buffer? Design an experiment that would determine how much acid or base could be added to your buffer. Give detailed instructions and show the results of your experiment.

5. In your report, fully describe the theory of buffers and how they work. Use relevant equations and explain the parts of each equation.

Calibration of pH electrode:
Always keep your pH electrode submerged in solution and be careful with it, as they are very fragile.
1. Open the microlab software by double clicking on the "Microlab" icon on the Desktop.
2. From the "Choose an Experiment" window, double click on "Microlab Experiment" icon.
3. From the "Data Sources" pulldown, select "Add a Sensor", and choose" pH/D.O." as the sensor.
4. Click on the red box surrounding the BNC cable input on the microlabunit picture.
5. In the "Choose a sensor" box, click pH and then click "Next".
6. In the "Calibration" window, click "Perform New Calibration".
7. In the "Calibrate a sensor" window, click "Add Calibration Point".
8. Rinse the pH electrode with DI water and submerge it in the pH = 4 buffer solution.
9. Once the reading has stabilized, enter 4 in the "Actual value" field and click OK.
10. In the "Calibrate a sensor" window, click "Add Calibration Point" again.
11. Rinse the pH electrode with DI water and submerge it in the pH = 7 buffer solution.
12. Once the reading has stabilized, enter 7 in the "Actual value" field and click OK.
13. Rinse the pH electrode with DI water and submerge it in the pH = 10buffer solution.
14. Once the reading has stabilized, enter 10 in the "Actual value" field and click OK.
15. Select "First Order (Linear)" from the "Curve Fit Choices" box, then click "Accept and Save this calibration" and click OK.
16. Save the file with a name specific the date of the lab such aspH_11_05_09", then click "Finish”.
17. Drag the pH sensor icon to empty box in the lower right of the micro lab screen to display the pH.

Unless otherwise stated, the content of this page is licensed under Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License