Question

Set the spectrophotometer to \(260 \mathrm{~nm}\) wavelength. Follow the instructions for your UV spectrophotometer to check the absorbance of the extracted DNA, and record on the Data Sheet.

Short answer

Set spectrophotometer to \(260 \mathrm{~nm}\), insert DNA sample, and measure absorbance.

Step-by-step solution

- Turn on the Spectrophotometer

Power on the UV spectrophotometer by pressing the power button. Allow it to warm up as per the manufacturer's instructions, usually for around 15 minutes.

- Set the Wavelength

Adjust the wavelength setting on the spectrophotometer to \(260 \mathrm{~nm}\). This can typically be done by selecting the 'Wavelength' option and inputting \(260\).

- Prepare the DNA Sample

Place your extracted DNA sample into a clean quartz cuvette. Make sure the cuvette is free from fingerprints and any contaminants that might affect the reading.

- Insert the Cuvette

Insert the cuvette containing the DNA sample into the spectrophotometer's sample holder, ensuring it is placed correctly as per the device's instructions.

- Measure Absorbance

Close the lid of the spectrophotometer and press the 'Measure' or 'Start' button to take the absorbance reading at \(260 \mathrm{~nm}\). Record the displayed absorbance value onto the Data Sheet.

Key concepts

UV spectrophotometer

A UV spectrophotometer is an essential piece of laboratory equipment used in molecular biology. It measures the absorbance and transmittance of UV light by a sample, providing valuable information about the sample's properties. For DNA analysis, UV spectrophotometers help determine the purity and concentration by measuring how much UV light the DNA absorbs. You need to turn on the spectrophotometer and allow it to warm up for about 15 minutes by pressing the power button and following the manufacturer's instructions. This ensures the instrument will give accurate readings.

DNA absorbance

DNA absorbance refers to the amount of UV light absorbed by DNA molecules in a sample. DNA absorbs UV light efficiently due to the presence of conjugated double bonds in the nucleotide bases. The key wavelength for measuring DNA absorbance is 260 nm because DNA shows maximum absorption at this wavelength. By comparing the absorbance value of the sample with known values, one can deduce the concentration and purity of the DNA. A high absorbance indicates a high concentration of DNA, while additional peaks at other wavelengths might indicate the presence of impurities.

260 nm wavelength

The 260 nm wavelength is crucial for DNA analysis since DNA molecules have their maximum absorbance at this specific wavelength. During spectrophotometric analysis, setting the spectrophotometer to a wavelength of 260 nm allows for an accurate measurement of DNA concentration. To set the wavelength, select the 'Wavelength' option on your spectrophotometer and input 260. The spectrophotometer will then use a specific monochromator to filter light so only light of 260 nm wavelength passes through the sample.

Sample preparation

Proper sample preparation is critical for accurate spectrophotometric analysis. Begin by placing your extracted DNA sample into a clean quartz cuvette—quartz is used because it does not interfere with the UV light. Ensure that the cuvette is free from fingerprints and contaminants, as these can skew the absorbance readings. Use a soft, lint-free cloth to wipe the cuvette if necessary. Insert the prepared cuvette into the spectrophotometer's sample holder, making sure it is positioned correctly according to the device's instructions.

Data recording

Once the sample is ready and the spectrophotometer is set to 260 nm, you can proceed to measure the absorbance. Close the lid of the spectrophotometer and press the 'Measure' or 'Start' button to begin the analysis. The spectrophotometer will measure how much UV light is absorbed by the DNA sample and display this value on the screen. Record the absorbance value accurately onto your data sheet, as this information is crucial for further calculations, such as determining DNA concentration and purity.