Introduction to Spectrophotometry

Objective:

To learn how to use a spectrophotometer.

Materials:

UV/visible spectrophotometer TruView Cuvette Polystyrene cuvettes

Background

Biophotonics is the study of the interaction of biological materials with light and other forms of radiant energy. Radiation is energy that comes from a source and can travel through material or space. The following illustration is of the electromagnetic spectrum along with various wavelengths.

1 Tissue and cells are composed of a variety of organic compounds (DNA, proteins, lipids, and carbohydrates). Light interacts with these molecules in different ways: reflection, absorption, transmission, and scattering. All biophotonic applications involve a light source that is passed through a target material and a detection sensor that reads the light emission from the material. A spectrophotometer has a light source that generates specific wavelengths. The light path passes through the cuvette, is absorbed by the material in the cuvette, and is read by a detector. The wavelength which the spectrophotometer is set to is referred to as the wavelength of absorbance. For example, a bacterium absorbs light at a wavelength of 600nm (A600). The amount of absorbed light per thickness of the sample is referred to as the Optical Density (OD).

There are several different types of spectrophotometers available. We will use a UV/Visible spectrophotometer to measure the concentration of organic compounds. More specifically, we will measure the concentration of bacteria and DNA. UV/visible Spectroscopy are based on the absorption of light. This spectrophotometer uses a Xenon flash lamp which emits both UV and visible wavelengths. The path of light in the spectrophotometer is as follows:

Part I – Measuring bacterial growth

Monitoring bacterial growth and harvesting a culture at the right moment is an important first step for any experiment which requires bacteria as a model organism. Bacterial growth occurs in four stages. The first phase, the lag phase, begins when the bacteria is added to the media. During this phase, bacterial growth is slow as the bacteria acclimate to the nutrients and the new environment. The second phase is called the log or exponential phase. It is during this phase in which the bacteria are reproducing at their maximum rate. The third phase is called the saturation or stationary phase. The growth rate of the bacteria slows during this phase due to the depletion of nutrients and the accumulation of waste products. The fourth and final phase is called the death phase. It is at this point that the bacteria begin to run out of nutrients and die.

There are several techniques which can be utilized in order to measure bacterial growth. The first technique involves using a Hemocytometer. A Hemocytometer is used to count the number of cells in a given volume. The problem with using a Hemocytometer is that the total number of bacteria is determined not the number of bacteria which are alive. The second method used to determine bacterial concentration is serial dilution. A cell culture is diluted several times and plated. The number of colonies and the dilution factor are used in order to determine the concentration of the bacteria.

The third technique involves using a spectrophotometer. The amount of light absorbed by the bacterial culture is measured. A wavelength of 600nm (A600) is used for measuring bacterial concentration. The benefit of using a spectrophotometer is that it’s quick and easy. The harvesting of a culture should be completed during the early log phase of cell growth. When measuring the rate of growth of bacteria in culture, an OD of .5-.7 indicates that the bacteria are in the early to mid log phase of growth. It is best to harvest the cells during this phase since the bacteria are at their peak rate of growth.

Protocol:

1. Obtain an overnight culture

2. Add 1ml of each overnight culture to a polystyrene cuvette.

4. Add 1ml of fresh media to a second cuvette (Used to zero the spectrophotometer)

5. Turn on the spectrophotometer.

6. Choose the bacteria concentration assay by pressing the OD600 button.

7. Press the “Enter” button for “Yes” when the screen prompts “A600 1.0=5.0e8cells/ml”

8. Place the cuvette with the fresh media into the sampling chamber.

9. Press the “Read Blank” button.

10. Remove the cuvette from the sampling chamber, and press the right arrow key to continue.

11. Place your first cuvette with culture into the sampling chamber and press the “Read Sample” button.

12. Press the left arrow button to exit the assay.

13. Press “cancel” and print out your results.

Analysis:

1. Record your results in your lab notebook

2. Based on your OD readings approximately which phase of growth were your samples in? (0.5-0.7 indicates early to mid log phase of growth)

5 Part II – Measuring DNA Concentration and Purity

Both the purity and concentration may contribute to the success or failure of an experiment such as a bacterial transformation or mammalian cell transfection. For this experiment you will first determine the purity of a sample of DNA and then determine the sample concentration (ug/ml).

A purity ratio is often used to determine the relative purity of a sample of DNA or RNA. DNA and RNA both absorb at a wavelength of 260nm while contaminants in the sample, such as proteins, absorb at a wavelength of 280nm. A ratio of the readings at A260 and A280 provides information on the relative purity of the DNA sample. An A260/A280 reading of 1.8 indicates a pure DNA sample while a reading of 2.0 indicates a pure RNA sample.

The concentration of a DNA or RNA sample can also be estimated using a spectrophotometer. The following calculation can be used to determine the concentration of a sample of DNA or RNA

Concentration = A260 * Conversion Factor * Dilution Factor

The following conversion factors should be used when using this formula:

Sample A260 of 1.0 OD

dsDNA 50.0 ug/ml ssDNA 37.0 ug/ml RNA 40.0 ug/ml

It is important to use a quartz cuvette when measuring RNA or DNA concentrations. Glass and plastic cuvettes absorb UV wavelengths, which limits their usefulness.

Protocol:

1. Obtain a sample of mini-prep DNA

2. Obtain two TruView cuvetted

3. Fill one cuvette with sterile water. This will be used as your blank

4. In the other culture tube, complete a 1:10 dilution of the stock DNA so that there is a volume of 60ul.

5. Turn on the spectrophotometer.

6. Choose the DNA/RNA assay.

7. Press the enter button to select dsDNA

8. Press the enter button when the screen reds “A260 1.0=50.0 ug/ml”

9. Press the dilution factor button and set the dilution factor to 5. Press enter.

10. Blank the spectrophotometer with the cuvette with the sterile water.

11. Place the cuvette with the diluted DNA into the sample chamber and press the “Read Sample” button.

12. Press the left arrow button to exit the assay. Press “cancel” and select to print out the results

Analysis:

1. What is the concentration of your DNA sample (ug/ul)?

2. What is the purity ratio of your sample? Is your DNA sample considered pure?