Spectrophotometer & Cuvettes

What are they and why do we use them? What types are there?

A spectrophotometer is used for measuring the light adsorption of a specific liquid. A cuvette is a specially designed vial for holding the liquid while it is measured by the spectrophotometer.

There are a wide variety of spectro’s available, but they can be generally sorted by;

1. Wavelength

   1. Visible

   2. Infrared

   3. UV

      1. Qubit and Nanodrop spectrophotometers fall into this category and have their own dedicated page.

2. Beam count

   1. Single beam

   2. Double beam

The most useful type of spectro for simple biotech work is a single beam, visible light spectrophotometer. This will allow you to measure the OD600 of cells.

It is very possible to build a DIY spectrophotometer. Multiple designs exist online, e.g. https://wiki.london.hackspace.org.uk/view/Project:OD600_Measurement

When do you use?

During protocols such as the induction of protein expression, or the creation of chemically competent cells - you will need to monitor the ongoing growth rate of a cell culture. This is typically done by measuring the absorbance of light with a wavelength of 600 nanometers.

How do you use?

OD600 Protocol:

Calibration will be machine specific, you should try to find an online protocol or video for your specific machine. The following steps should be generically true;

1. Prior to inoculation, remove a 1 ml sample of blank medium using sterile technique and add it to the cuvette.

2. Insert the cuvette (ensuring the beam can travel through the clear sides of the cuvette, not the rugged sides) into the spectrophotometer. Set the machine to read this as Adsorption = 0.

3. Inoculate your medium and add the flask/vial back to the incubator.

4. After two hours of incubation, remove another 1 ml of sample and add it to a fresh cuvette. Measure the adsorption. it should be around 0.1-0.2, but will vary based upon cell type.

5. Measure every 30 minutes until the OD600 is between 0.3-0.7

   1. Some protocols recommend exactly 0.5.

6. You can now induce your cell culture for protein expression, or add it to ice to prepare for chemically competent cell preparation.