2.2: Using the spectrophotometer to evaluate your pipetting skills
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Since you will be using micropipettes for all of your experiments, the quality of your results will depend on proper operation of the micropipette. Today’s laboratory will lead you through some exercises that will show you how to use micropipettes correctly and point out some common pitfalls associated with their use. Your results will also provide information about whether the pipettes are functioning properly.
In these exercises, you will be using the spectrophotometer to determine if your pipetting is accurate and precise. You will
be using micropipettes to combine various volumes of water and solutions of a blue dye, bromophenol blue (BPB). You will measure the absorbance of the resulting solutions at 590 nm (A590), which is close to the absorbance maximum of bromophenol blue at neutral pH. Measuring errors will be reflected in the spectrophotometer readings.
The spectrophotometer readings provide an indirect measurement of pipette performance. The proper way to calibrate the micropipettes would be to weigh out volumes of water, which
has a specific gravity of 1.0 g/mL. Unfortunately, we do not have enough balances with sufficient accuracy for the class to perform the measurements. If you suspect inaccuracies in the micropipettes that you are using, refer them to the teaching staff, who will test them properly.