1. An Introduction to the Spectrophotometer

2. Meet your Spectrophotometer Meet your spectrophotometer

3. You will Practice Calibration Calibration= assure equipment is accurate

4. 1.Set wavelength at 580nm 2.0% T 3.100% T Calibration wavelength

5. 3. Pipet 4 ml of water into cuvette 4. Place cuvette into spectophotometer

6. 5. Close lid 6. Set transmittance to 100%

7. Step 1: Make a standard curve of known concentrations Standard Curve

8. A graph that allows the correlation between a qualitative measurement such as absorbance and known concentrations. What is a standard curve?

9. HOW TO PREPARE YOUR PRACTICE STANDARD CURVE How to use the “pipetman” How to make dilutions

11. Push plunger to “first stop” Place tip in solution Aspirate sample by releasing plunger

12. HOW TO PREPARE YOUR PRACTICE STANDARD CURVE Abs. Concentration (independent) (dependent) What you manipulate What you measure

13. How to use your standard curve:

14. (independent variable) (dependent variable) Absorbance concentration 0.2.4.6.8 1.0.2.2.4.4.6.8.81.0 0 Unknowns Abs. 0.77 Abs. 0.40 X X X Sample graph to calculate unknown concentrations

15. Example of data & standard curve

16. DNA ug/ml 0.0 0.2 0.4 0.6 0.8 1.0 Absorbance 260 nm 0.18 0.35 0.60 0.70 0.95 0.0 Sample DATA (example only!) Concentration

17. (independent variable) (dependent variable) Absorbance (260nm) DNA (ug/ml) 0.2.2.4.6.8 1.0.2.4.6.81.0 0 Sample graph from sample data Low sensitivity Values>1.0Abs

18. ENZYME LAB Next class: Apply principles to

19. Preview of things to come: Important Terms Enzyme Substrate Product Standard Curve

20. Next Week Experiment: To study the affect of various parameters on enzyme activity by measuring product formation –A. p-nitroaniline Standard curve –B. of substrate on product formation over time –C. of temperature on product formation over time –D. of pH on product formation over time

21. Substrate Products Enzyme Active Site Enzyme Binding of Substrate to Catalyst