Lab #1: Pippeting, Buffers, Serial Dilutions and Spectrophotometry

Background 

As presented in the lecture, the science of spectrophotometry is the backbone of most enzyme linked metabolite assays.  Lecture content also revealed the primary role of Beer's Law in this scientific approach at quantifying metabolite concentrations.  This laboratory provides you with experience in pippeting, developing serial dilutions, and performing computations based on Beer's Law and a standard curve to solve for the concentration of an unknown solution of NADH.

1. Using the known NADH solution, develop a serial dilution that spans the absorbance range from 0 to 1.5 (or whatever the absorbance value is for the stock NADH solution).  You will need to test specific dilutions to assess where this range lies relative to the concentration of the standard (stock) NADH solution.
2. For every known concentration that forms your serial dilution standard curve, also compute the NADH concentration at 340 nm using Beer's Law.  These should be almost identical to your serial dilutions based on computations from the standard solution.
3. Report the two methods of concentrations determination in a table or graph.
4. Explain any discrepancies.

Unknown Solution

1. Solve for the unknown concentration of the NADH solution based on what you have learned from the work above.

2. Once again, present data for your procedures and solutions based on Beer's Law and your standard curve.

Spectrophotometer Use

Make sure you allow 20 min of warm up time.  The default wavelength for the spec is 340 nm, so you do not have to change anything.  I will show you how to use the instrument in class, as well as set-up the unit for zeroing to distilled water as a blank, etc.

References: use any biochemistry or spectrophotometry texts.  Note: there are some good reference enzymatic biochemistry texts in the Biochem lab.