Lab Techniques

• Hazards that could be encountered in the lab include toxic and corrosive chemicals, heat and flammable substances, mechanical equipment and pathogenic organsims
• We can identify hazards and implement control measures to minimise risks and reduce the number of hazards by creating a risk assessment.
• A risk is the likelihood of a hazard causing harm.
• Control measures include appropriate handling and disposal techniques, protective clothing and equipment, and aseptic techniques
• Linear dilutions differ by equal interval and are made up to the same overall volume, producing different concentrations of the stock solution.
• Log dilutions differ by constant proportion, and each dilution acts as a stock solution for the next dilution.
• Log dilutions are often used in microbiology to help estimate the density of cells in a solution.
• To make up solutions of varying concentrations from stock solutions, use the formula: C1V1=C2V2.
• V1 is the volume of the stock solution
• C1 is the concentration of the stock solution
• V2 is the volume of the diluted solution
• C2 is the concentration of the diluted solution
• Colorimetry can estimate the concentration of a known solute by illuminating a small sample of the test substance using a coloured light source.
• The colorimetry sample is held in a transparent cuvette.
• In colorimetry, different light colours are used depending on the wavelength which will be absorbed the most by the sample.
• In colorimetry, the level of absorbance is measured electronically, and this is used to estimate concentration.
• Colorimetry can be used to estimate the density of cells in a culture and the turbidity of a liquid as it also measures transmission.
• For each colorimetry experiment, the machine is calibrated using a blank cuvette containing only the solvent. This acts as a control value for comparison.
• Using linear dilutions and a colorimeter we can produce a standard curve.
• A buffer is a solution where the pH changes very little when small volumes of acid or alkali are added.
• In laboratory experiments, buffers can be selected so that the pH of solutions can be controlled.
• In cell culture media, buffers are used to prevent pH changes that could occur as a result of the build-up of waste products.
• A centrifuge can be used to separate molecules based on density.
• Centrifugation involves spinning a sample at a very high speed, causing more dense material to accumulate at the bottom of the tube, and the less dense materials to come to the top
• More dense materials at the bottom of the tube are known as the pellet, and less dense components at the top are known as the supernatant.
• Paper and thin layer chromatography is used to separate sugars and amino acids.
• The speed that each solute travels along the chromatogram depends on its differing solubility in the solvent used. More soluble makes the solute travel faster, and therefore further.
• Paper chromatography uses chromatography paper, and thin layer involves the use of silica gel or solid cellulose on a glass slide.
• Affinity chromatography is used to separate one specific protein from a mixture.
• In affinity chromatography, a solid matrix or gel column with specific receptor molecules attached is used. As the mixture passes through the column, target proteins with a high affinity to these molecules will bind. Non-target proteins with a low affinity to these molecules will be washed out.
• Gel electrophoresis is a separation technique that can be used on charged molecules such as DNA and nucleic acids.
• In gel electrophoresis, molecules are run through an agarose gel matrix with an electric current applied to it.
• In gel electrophoresis, the largest molecules move the slowest, so are found closest to the starting point, whereas smaller molecules move much quicker, so end up further away.
• Native gels do not denature the molecules, separation is by shape, size and charge. The overall charge of the protein will also effect the rate at which it travels.
• In SDS-page, a compound is added to the sample which denatures proteins, and makes them linear. All molecules are given an equally negative charge, and proteins are separated by size alone.
• The isoeletric point of a protein is the pH at which it has no net charge.
• At the isoelectric point, the pH will precipitate out of solution