Laboratory Techniques for Biologists Flashcards
(30 cards)
What is a hazard ?
A hazard is anything which could harm
What can present a hazard ?
Substances, organisms, and equipment in a laboratory can present a hazard, including;
-Toxic or corrosive chemicals
-Heat or flammable substances
-Pathogenic organisms
-Mechanical equipment
What is risk ?
Risk is the likelihood of harm arising from exposure to a hazard.
What is a risk assessment ?
Risk assessment involves identifying control measures to minimise the risk.
What are control measures of a risk ?
Control measures are any measures taken to eliminate or reduce the risk of injury or bodily harm.
Control measures include using;
-appropriate handling techniques
-protective clothing and equipment (Safety goggles, lab coats, gloves etc)
-aseptic technique
What is and what are types of dilution ?
-A dilution is taking an original solution and making the concentration weaker.
-Includes linear and log dilutions.
What is a linear dilution ?
Dilutions in a linear dilution series differ by an equal interval, for example 0·1, 0·2, 0·3 and so on.
What is a log dilution ?
Dilutions in a log dilution series differ by a constant proportion, for example 10-1, 10-2, 10-3 and so on.
How can the production of a standard curve be used to determine an unknown ?
Plotting measured values for known concentrations to produce a line or curve allows the concentration of an unknown to be determined from the standard curve.
How are buffers used to control pH
Addition of acid or alkali has very small effects on the pH of a buffer, allowing the pH of a reaction mixture to be kept constant.
Describe the method and uses of a colorimeter to quantify concentration and turbidity
Calibration with appropriate blank as a baseline;
-Use of absorbance to determine concentration of a coloured solution using suitable wavelength filters, such as haemoglobin in blood.
-Use of percentage transmission of light to determine turbidity, such as cells in suspension.
Turbidity = the quality of being cloudy, opaque, or thick with suspended
What are the separation techniques ?
1) Centrifugation
2) Paper, thin-layer, and affinity chromatography
3) Gel electrophoresis
5) Iso-electric points
4) Separation of proteins
What is a centrifuge used for ?
-Separates substances of different densities
-More dense components settle in the pellet; less dense components remain in the supernatant
What is paper and thin layer chromatography used for ?
-Can be used for separating different substances such as amino acids and sugars
-The speed that each solute travels along the chromatogram depends on its differing solubility in the solvent used.
What is affinity chromatography used for ?
-Separating proteins
1) A solid matrix or gel column is created with specific molecules bound to the matrix or gel.
2) Soluble, target proteins in a mixture, with a high affinity for these molecules, become attached to them as the mixture passes down the column.
3) Other non-target molecules with a weaker affinity are washed out.
What is gel electrophoresis ?
-Used to separate proteins and nucleic acids.
-Charged macromolecules move through an electric field applied to a gel matrix (will move towards positive electrode)
-Native gel and SDS-PAGE
What is native gel electrophoresis ?
Native gels separate proteins by their shape, size and charge
Native gels do not denature the molecule so that separation is by shape, size and charge.
What is SDS-PAGE
SDS–PAGE separates proteins by size alone
SDS–PAGE gives all the molecules an equally negative charge and denatures them, separating proteins by size alone.
What are Isoelectric points ?
-IEP is the pH at which a soluble protein has no net charge and will precipitate out of solution.
-Can be used to separate proteins in a mixture, If the solution is buffered to a specific pH, only the protein(s) that have an IEP of that pH will precipitate
-Can also be used in electrophoresis
Describe isoelectric points in electrophoresis
-Proteins can also be separated using their IEPs in electrophoresis.
-Soluble proteins can be separated using an electric field and a pH gradient. A protein stops migrating through the gel at its IEP in the pH gradient because it has no net charge.
What are immunoassay techniques ?
-Immunoassay techniques use antibodies to detect and identify specific proteins.
-Antibodies can be used to detect both the presence and concentration of a protein within a solution.
-They rely on the specificity of antibodies; their ability to recognise and bind with only one protein.
(The thing we are trying to detect is called the “antigen”)
How do immunoassay techniques work ?
-These techniques use stocks of antibodies with the same specificity, known as monoclonal antibodies.
-An antibody specific to the protein antigen is linked to a chemical ‘label’. The ‘label’ is often a reporter enzyme
producing a colour change, but chemiluminescence, fluorescence and other reporters can be used.
-In some cases the assay uses a specific antigen to detect the presence of antibodies.
What is Western Blotting ?
Western blotting is a technique, used after SDS–PAGE electrophoresis.
The separated proteins from the gel are transferred (blotted) onto a solid medium. The proteins can be identified using specific antibodies that have reporter enzymes attached.
What is microscopy ?
-Bright-field microscopy is commonly used to observe whole organisms, parts of organisms, thin sections of dissected tissue or individual cells.
-Fluorescence microscopy uses specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues.
