Midterm Prep PDF

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Lab 1 Introduction, lab math and cells a. Metric system prefixes i. Kilo- (k) = 103 ii. Centi- (c) = 10-2 iii. Milli- (m) = 10-3 iv. Micro- (μ) = 10-6 v. Nano- (n) = 10-9 vi. Pico- (p) = 10-12 b. Dilution and solution calculations i. Calculate different concentrations based on molarity and molecular weight ii. Calculate concentrations based on %w/v or v/v iii. Calculate dilutions based on stock solutions (C1V1=C2V2) c. Pipetman i. p1000 = 1000-100 microliters ii. p200 = 200-20 microliters iii. p20 = 20-0 microliters d. Order of sizes of cells, organelles, macromolecules, and small biological molecules i. Atom → small molecule → globular protein → ribosome → virus → bacterium → animal cell → plant cell e. Eukaryotic cell: organelles, structure, and functions Lab 2 Microscopy introduction a. DME compound microscope: parts and functions *look at 2A lab slide* i. Ocular lens (eyepiece) = provides a small part of the total magnification and establishes the size of the field of view ii. Objective lens = collects a cone of light rays to form the image that you see, provides most of the magnification and overall resolution of the image iii. Condenser = focuses a cone of light rays onto the specimen on the slide iv. Field iris aperture = located at the base of the microscope at the point where the light that will form the image first enters the system b. Kohler illumination i. Alignment of the condenser and objective lenses that maximize resolution, illuminate the image evenly, reduces reflection and glare, and minimizes heating ii. Works by focusing the beam path while the image is defocused c. Magnification, resolution and depth of field i. Magnification = the degree to which an image is enlarged ii. Resolution (resolving power) = shortest distance between two points in a specimen that can still be distinguished by the observer as separate entities, level of detail in the image 1. Dependent on wavelength of light, numerical aperture (NA =nsin(theta)) a. N = refractive index of the glass lens b. Theta = angle of incidence of the light onto the lens 2. Best resolution = when d (=0.61(wavelength/NA)) is small → NA high iii. Depth of field = thickness of the specimen that is in focus at one time, dependent on NA and the total magnification

1. Decreases as magnification and NA increase iv. Maximum diameter of field view = field #/ objective lens magnification d. Tetrahymena cell: organelles, structure, and functions i. Basal bodies 1. Anchor cilia ii. Cilia 1. Promotes cell movement iii. Cytoproct 1. Waste remaining in vacuoles is exocytosed iv. Macronucleus 1. Somatic nucleus v. Micronucleus 1. Germline nucleus vi. Oral apparatus (mouth) 1. Cilia direct food into oral apparatus and then food goes to vacuoles vii. Vacuoles 1. Contractile vacuole = collects water and expels it for osmotic regulation 2. Food vacuole = food particles phagocytosed at oral apparatus Lab 3 Tetrahymena and microscopy a. Types of lenses and oil immersion i. 10X, 40X, 100X (can do oil immersion) b. Phase contrast i. Constructive and destructive interference of light waves that recombine to form the image ii. Annulus slot placed below the condenser lens and additional annulus in objective lens c. Stereo-microscope i. Low magnification observation d. Inverted scope i. Light source and condenser on top; able to see specimen in natural environments, not plated e. Tetrahymena thermophila = model organism i. Cheap, easy to use; grows fast at room temp; large (30-50 micrometers); easy genetics; lots of cilia (cellular motility); complex conjugation involved programmed nuclear death (PND = apoptosis and autophagy) ii. Log phase = conditions of plentiful food 1. Binary fission = MIC replicates and chromosomes separate normally by mitosis; in the MACs the multiple chromosomes randomly segregate (amitosis) 2. Generation time (double time) is 3-4 hours iii. Starvation 1. Cell shrinks but nucleus, oral structure, and contractile vacuole remain the same f. Micro and macronucleus of Tetrahymena thermophila i. Micro = germline and has 5 pairs of chromosomes, transcriptionally silent ii. Macro = somatic genome and has ~200 chromosomes with a ploidy of ~45 during G1 phase, transcriptionally active

g. Conjugation of Tetrahymena thermophila i. After starvation conjugation occurs between 2 starved cells of different mating types (7 mating types) ii. Micronucleus undergoes meiosis to form 4 haploid nuclei; 1 migrates to the anterior end of the cell and the other 3 are degraded iii. Surviving haploid nucleus undergoes mitosis and each cell of the mating pair exchanges 1 nucleus with the other; haploid nuclei fuse and make diploid nucleus iv. Diploid nucleus undergoes 2 rounds of mitosis and 2 nuclei move to anterior and turn into Macronuclei v. 1 MIC at the posterior end is degraded; with food, the MIC undergoes another round of mitosis vi. Old MAC degraded by PND, the cells then separate and divide vii. START = 2 starved cells; END = 4 vegetative cells Lab 4 Bioinformatics a. Eukaryotic gene b. Gene expression c. Gene malfunction d. BLAST and E-Value i. BLAST = % identity of amino acids ii. E-value = expected number of times that the given BLAST score would appear when searching a random database of the given size; affected by database size, length of query, gap size, and placement e. Protein domain i. Part of the protein sequence and structure that can evolve, function, and exist independently of the rest of the protein chain and may appear in a variety of evolutionarily related proteins ii. Each domain forms a compact 3D structure and can be independently stable and folded (25-500 amino acids) f. TTHERM# i. Identifies a unique gene g. How does the T. thermophila genetic code (codon table) differ from the Universal genetic code? i. Only 1 stop codon Lab 5 Primer design and PCR theory a. Apoptosis: purpose, mechanism, associated diseases i. Cell shrinks and dies without leaking contents into the surrounding area ii. Caspases (proteases) activate and degrade cellular proteins, nuclear lamins and cytoskeleton 1. Caspases = cysteine proteases 2. Initiator caspases activate the pathways leading to cell death a. Activation of kinases → break cell adhesions and inhibits cell survival pathways

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b. Cleaving lamins → nuclear envelope breaks down and cytoskeletal proteins → cell shape changes c. Activating endonuclease (CAD) → DNA is fragmented iii. Normal mechanism during development; reduced or elevated apoptosis linked to disease PCR: reagents, process, purpose i. Reagents = DNA template, primers, MgCl2, dNTPs, Taq DNA polymerase, reaction buffer ii. Process = denaturation (95) → annealing (65-69) → elongation (72) iii. Purpose = amplify specific DNA sequence Primer design rules i. 5’ → 3’ direction; 17-28 nt long and amplify a DNA sequence 300-1000 bp long ii. Forward primer in the same direction as the template DNA sequence; Reverse primer in the opposite direction as the template DNA sequence iii. Approx 50% GC content; Melting temperature around 55 iv. Primer dimers = intermolecular complementary; hairpins = intramolecular complementarity v. GC clamp = primer terminal ends in G or C 1. G/C base pairs more stable than AT base pairs Predicting PCR product sizes from cDNA and gDNA Gene Expression

Lab 6 DNA preps and performing PCR a. Genomic DNA prep: steps and purpose i. Lyse cells in TRI reagent (acidic so RNA remains in aqueous phase) ii. Add chloroform and spin to separate layers iii. Pipette off top layer (RNA will be in aqueous solution) iv. Add isopropanol to precipitate the RNA v. Spin RNA and remove supernatant vi. Wash RNA pellet twice with 75% ethanol and dissolve in water b. If you want to measure gene expression using PCR, why do you have to use CDNA and not gDNA? i. cDNA only has protein coding sequences (only exons) c. Reverse Transcription (RNA to cDNA) i. RNA cannot directly be the genetic material for PCR reactions → too unstable Lab 7 Agarose gels and more PCR a. Analyze PCR products/ interpret agarose gels i. DNA runs from (-) to (+) electrode; Shorter DNA runs further ii. More concentrated agarose gels will have a smaller DNA size resolution in kbp b. Preparing agarose gels i. To make a 1.5% w/v agarose gel multiply 0.015(volume of TAE buffer used) c. Understand the role that ATG4 (chosen gene) plays in autophagy or apoptosis i. Factor in the Atg 8 conjugation system 1. Exposes a glycine residue at the c-terminus of Atg 8

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Cleaves Atg8-PE (delipidation/deconjugation) 1. Recycles Atg 8 for the next rxn 2. Promotes elongation step of isolation membrane IMPORTANT FOR AUTOPHAGOSOME FORMATION 1. Inhibition of Atg 4 leads to inhibition of autophagy at autophagosome production

Autophagy and Apoptosis a. Mechanism of autophagy and where it occurs in endomembrane system of cell i. Co-operation with ubiquitin-proteasome system (UPS) → regulates protein degradation and organelle turnover ii. Atg proteins (6 major groups) recruited in hierarchical manner to expand the membrane and produce the autophagosome; then fuses with lysosome and release contents for degradation iii. Occur in the constitutive secretion area of the endomembrane system b. Purpose of autophagy and diseases that occur due to malfunctions in the mechanism i. Stress-induced self-preservation adaptation that avoids cell death by breaking down organelles, proteins, and lipids to provide materials for cells to build new molecules ii. Occurs all the time at basal rates to remove damaged organelles and mis-folded proteins; Stimulated by oxidative stress, environmental stress, starvation, and infection iii. Three Types 1. Macroautophagy = degrades old or damaged organelles or protein aggregates too large for the ubiquitin-proteasome system; phagophores → autophagosomes → bind with lysosome 2. Microautophagy = lysosomes directly engulf cytoplasmic contents 3. Chaperone-mediated autophagy = chaperone heat-shock-like proteins bind specific proteins and enable them to be transported into lysosomes c. Programmed nuclear death in T. thermophila--role of autophagy and apoptosis i. MAC degradation and 3 MIC haploid after meiosis d. Purpose and function of mTOR in autophagy i. mTOR = kinase active when cells are unstressed; phosphorylates Agt 13 ii. Inhibition of mTOR initiates autophagy in mammalian cells e. Mechanism of apoptosis and enzymes involved i. Leads to programmed cell death where organelles are destroyed, DNA is fragmented and cells shrink and die ii. caspases f. Intrinsic vs. extrinsic pathway i. Intrinsic = internal signals, something wack with DNA; triggered when cell is stressed 1. Regulated by Bcl-2 protein family; caused by an imbalance of anti- and pro-apoptotic proteins 2. Pro-apoptotic proteins make a channel in the outer mitochondrial membrane so that mitochondrial proteins (CYTOCHROME C) leak out and form an apoptosome which activates initiator caspase 9 which then activates executioner caspases to promote apoptosis ii. Extrinsic = external signals (cytokines released by immune system, chemo, radiation)

1. Tumor Necrosis Factor -- protein which activates pathway and recruits procaspase 8 which then activates executioner caspases for cellular apoptotic destruction g. Purpose of apoptosis and diseases that occur due to malfunctions in the mechanism h. CELL BIO LAB GENERAL RESEARCH FOCUS Lab 9 1. How to optimize PCR a. Clean workspace so the PCR isn’t contaminated b. DNA quality and quantity-- template needs to be pure and there needs to be the correct amount c. Optimal annealing temperature i. Depends on the composition and the length of the sequence ii. Within 5 °C of the primer melting temperature iii. Melting temperature is defined as when 50% of the strand will be annealed iv. Higher the GC content, the higher the melting temperature v. Temp= 2(A+T) + 4(G+C) vi. If the temperature is too low, there will be nonspecific PCR (multiple bands) vii. If temperature is too high, there will be no annealing viii. Run a temperature gradient d. Magnesium concentration i. Too little magnesium-- no bands (cofactor with Taq polymerase) ii. Too much magnesium-- incorrect annealing (multiple bands) iii. Try at different increments to see the ideal concentration e. PCR time i. The longer the stand, the longer time it needs 2. 3 kinds of scientific misconduct a. Fabrication- making data up b. Falsification- changing data or omitting results c. Plagiarism- taking someone else’s work Lab 10 1. qPCR a. Normal PCR- analyzed after reaction completion b. qPCR- monitors DNA amplification as the reaction continues i. Monitored by the increase of fluorescent dye ii. Brighter dye when strands of DNA bind c. Output graph of qPCR (like an exponential curve) i. Lag (beginning), exponential (middle), linear, plateau (end) ii. Lag- not detectable iii. Exponential- where it begins to be detected iv. Cq values can be determined to compare more than one sample...