Lab techniques
62 flashcards covering Lab techniques for the MCAT Biology & Biochemistry section.
Lab techniques in biology and biochemistry refer to the hands-on methods scientists use to conduct experiments, analyze samples, and gather data. These include practices like microscopy for observing cells, centrifugation for separating substances, electrophoresis for sorting molecules, and PCR for amplifying DNA. Essentially, they provide the tools to manipulate and study biological materials, helping researchers uncover how living systems work at the molecular and cellular levels. Mastering these techniques is crucial because they form the foundation of real-world lab work and scientific inquiry.
On the MCAT, lab techniques appear in Biology and Biochemistry questions that test your ability to identify procedures, interpret experimental results, and evaluate experimental design. You might encounter multiple-choice questions asking about the purpose of a technique, potential errors like contamination, or how to troubleshoot issues. Common traps include confusing similar methods, such as gel electrophoresis with chromatography, or overlooking safety protocols. Focus on understanding applications, data analysis, and the logic behind each technique to handle these questions effectively.
A concrete tip: Practice sketching simple experimental setups to visualize how techniques interconnect.
Terms (62)
- 01
Light Microscopy
A technique using visible light and lenses to magnify small objects, allowing visualization of cells and tissues up to about 1000x magnification.
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Electron Microscopy
A method using a beam of electrons to create high-resolution images of very small structures, such as viruses or cellular organelles, with magnifications up to 500,000x.
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Phase Contrast Microscopy
A light microscopy technique that enhances contrast in transparent specimens by exploiting differences in refractive indices, useful for observing live cells without staining.
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Fluorescence Microscopy
A technique where fluorescent dyes or proteins are used to label specific molecules, allowing visualization of their location and movement within cells under specific light wavelengths.
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Resolution in Microscopy
The ability of a microscope to distinguish two closely spaced objects as separate, determined by the wavelength of light or electrons used and the numerical aperture of the lens.
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Magnification
The process of enlarging the apparent size of an object through lenses, calculated as the product of the objective and eyepiece magnifications in optical microscopes.
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Centrifugation
A technique that uses centrifugal force to separate mixtures based on density, commonly employed to isolate cellular components like nuclei or mitochondria from a homogenate.
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Differential Centrifugation
A stepwise centrifugation process that separates cellular components by applying increasing speeds, allowing heavier particles to pellet first while lighter ones remain in suspension.
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Density Gradient Centrifugation
A centrifugation method where samples are layered over a gradient of increasing density, separating particles based on their buoyant density for purification of organelles or viruses.
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Sedimentation Coefficient
A measure of how quickly a particle sediments in a centrifuge, expressed in Svedberg units, which depends on the particle's size, shape, and density relative to the medium.
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Paper Chromatography
A technique that separates mixtures of compounds based on their affinity for a stationary paper phase and a mobile solvent phase, often used to analyze amino acids or plant pigments.
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Thin-Layer Chromatography
A chromatography method using a thin layer of adsorbent material on a plate to separate compounds based on their partitioning between the stationary and mobile phases, faster than paper chromatography.
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Gas Chromatography
A technique that vaporizes a sample and separates its components as they pass through a column with a carrier gas, based on their volatility and interaction with the stationary phase.
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High-Performance Liquid Chromatography
An advanced liquid chromatography method that uses high pressure to force a solvent through a column, separating compounds by their affinity and size for analytical or preparative purposes.
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Rf Value
In chromatography, the ratio of the distance traveled by a compound to the distance traveled by the solvent front, used to identify substances based on their relative mobility.
- 16
Gel Electrophoresis
A technique that separates macromolecules like DNA or proteins through a gel matrix under an electric field, based on size and charge, with smaller molecules migrating faster.
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SDS-PAGE
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a method that denatures proteins and separates them by molecular weight in a polyacrylamide gel, commonly used for protein analysis.
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Agarose Gel Electrophoresis
A form of gel electrophoresis using agarose as the matrix to separate nucleic acids like DNA fragments by size, visualized with stains under UV light.
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Isoelectric Focusing
An electrophoresis technique that separates proteins based on their isoelectric points in a pH gradient, where proteins migrate until they reach a pH matching their net charge of zero.
- 20
Beer-Lambert Law
The principle that absorbance of light by a solution is directly proportional to the concentration of the absorbing substance, path length, and its molar absorptivity, used in spectrophotometry.
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Spectrophotometry
A technique that measures the amount of light absorbed or transmitted by a sample at specific wavelengths, quantifying concentrations of substances like proteins or DNA.
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Absorbance and Concentration
In spectrophotometry, absorbance is linearly related to concentration via the Beer-Lambert law, allowing calculation of unknown concentrations from a standard curve.
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Polymerase Chain Reaction
A molecular technique that amplifies specific DNA sequences through repeated cycles of denaturation, annealing, and extension using DNA polymerase, essential for genetic analysis.
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Primers in PCR
Short, single-stranded DNA sequences that bind to the target DNA template, defining the region to be amplified and initiating synthesis by DNA polymerase.
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Taq Polymerase
A heat-stable DNA polymerase enzyme derived from Thermus aquaticus, used in PCR to withstand high temperatures during denaturation without losing activity.
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Annealing Temperature
The temperature in PCR cycles where primers bind to the template DNA, typically set a few degrees below the primers' melting temperature for optimal specificity.
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Western Blotting
A technique that detects specific proteins in a sample by separating them via gel electrophoresis, transferring to a membrane, and probing with antibodies.
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Southern Blotting
A method to detect specific DNA sequences by digesting DNA with restriction enzymes, separating fragments via gel electrophoresis, transferring to a membrane, and hybridizing with a probe.
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Northern Blotting
A technique similar to Southern blotting but used for RNA, separating RNA fragments by electrophoresis, transferring to a membrane, and detecting with a complementary probe.
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ELISA
Enzyme-linked immunosorbent assay, a technique that uses antibodies and enzymes to detect and quantify antigens or antibodies in a sample, common in medical diagnostics.
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Direct vs. Indirect ELISA
In direct ELISA, the primary antibody is directly labeled with an enzyme, while in indirect ELISA, a secondary antibody detects the primary one, amplifying the signal for greater sensitivity.
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Flow Cytometry
A technique that analyzes and sorts cells based on their physical and chemical characteristics, such as size and fluorescence, by passing them through a laser beam in a fluid stream.
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Fluorescence-Activated Cell Sorting
An extension of flow cytometry that physically separates cells based on their fluorescence properties, used for isolating specific cell populations in research.
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Gram Staining
A differential staining technique that classifies bacteria as Gram-positive or Gram-negative based on their cell wall properties, using crystal violet, iodine, alcohol, and safranin.
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Acid-Fast Staining
A staining method that identifies bacteria with high lipid content in their cell walls, like Mycobacterium, by resisting decolorization with acid-alcohol after primary staining.
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Microbial Culture Media
Nutrient-rich substances used to grow microorganisms in the lab, categorized as selective, differential, or enriched to isolate and identify specific bacterial species.
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Aseptic Technique
A set of practices to prevent contamination of cultures by unwanted microorganisms, including sterilization of equipment and working in a clean environment.
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Autoclaving
A sterilization method using high-pressure steam at 121°C to kill microorganisms and spores on lab equipment, ensuring a sterile environment for experiments.
- 39
Cuvette in Spectrophotometry
A small, transparent container used to hold the sample in a spectrophotometer, with precise path lengths to ensure accurate measurement of light absorption.
- 40
Blank Solution
A sample without the analyte, used in spectrophotometry to calibrate the instrument and subtract background absorbance for accurate readings.
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Enzyme Assay
A laboratory procedure to measure the activity of an enzyme by quantifying the rate of substrate conversion to product, often using spectrophotometry or colorimetry.
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Michaelis-Menten Kinetics
A model describing enzyme kinetics where reaction rate depends on substrate concentration, reaching a maximum velocity when the enzyme is saturated.
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Km and Vmax
In enzyme kinetics, Km is the substrate concentration at half of maximum velocity, indicating enzyme affinity, while Vmax is the maximum reaction rate achievable.
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Lineweaver-Burk Plot
A double-reciprocal plot of enzyme kinetics data that linearizes the Michaelis-Menten equation, used to determine Km and Vmax from the x-intercept and slope.
- 45
Restriction Digestion
The process of cutting DNA at specific sequences using restriction enzymes, essential for molecular cloning and genetic engineering techniques.
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Ligation in Cloning
The enzymatic joining of DNA fragments using DNA ligase, typically after restriction digestion, to create recombinant DNA molecules for cloning.
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Bacterial Transformation
The process by which bacteria take up foreign DNA from their environment, often facilitated by chemical treatment or electroporation, used in genetic engineering.
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Plasmid Vectors
Small, circular DNA molecules that replicate independently in bacteria and are used as vectors to carry and express foreign genes in cloning experiments.
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Common Trap: Pipetting Errors
In lab techniques, inaccurate volume measurements from improper pipetting can lead to erroneous results, so calibration and technique practice are crucial.
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Strategy: Avoiding Contamination
In molecular biology techniques like PCR, use separate areas for pre- and post-amplification, wear gloves, and use filter tips to prevent carryover of amplified DNA.
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Worked Example: PCR Cycle Calculation
For a 20-cycle PCR, starting with one DNA template, the final copies are 2^20, or about 1 million, illustrating exponential amplification for detecting rare sequences.
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Denaturing Agents
Chemicals like urea or SDS that disrupt hydrogen bonds and hydrophobic interactions in proteins or DNA, used in techniques like electrophoresis to unfold molecules.
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Buffers in Lab Techniques
Solutions that resist pH changes, essential in experiments like enzyme assays or electrophoresis to maintain optimal conditions for biological molecules.
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pH Indicators
Substances that change color at specific pH levels, used in titrations or to monitor pH in biochemical reactions for accurate experimental control.
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Titration Curve
A graph plotting pH against the volume of titrant added, showing the equivalence point in acid-base titrations, useful for determining protein pKa values.
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Dialysis
A technique that separates small molecules from larger ones by diffusion through a semi-permeable membrane, commonly used to remove salts from protein solutions.
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Ultrafiltration
A filtration method using membranes with specific pore sizes to separate macromolecules based on size, applied in concentrating proteins or purifying samples.
- 58
Chromatography Elution
The process of washing compounds through a chromatography column with a solvent, separating them based on their affinity for the stationary and mobile phases.
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Affinity Tags
Protein sequences added to recombinant proteins for purification, such as His-tags that bind to nickel columns in affinity chromatography.
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Protein Purification Steps
A series of techniques like centrifugation, chromatography, and electrophoresis to isolate a specific protein from a complex mixture based on its unique properties.
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Nucleic Acid Hybridization
The base-pairing of complementary nucleic acid strands, fundamental to techniques like blotting and PCR for detecting specific sequences.
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Radioactive Labeling
A method incorporating radioactive isotopes into molecules for detection in techniques like autoradiography, allowing visualization of DNA or proteins.