Practical Flashcards
Staining of Nucleic Acids
Methyl Green and Pyronin
DNA stained blue, RNA stained pink
Staining of Nucleic Acids/Polysaccharides
Toluidine Blue
Nucleic Acids stained purple, polysaccharides stained blue
Polarity of pancreatic acinar cell
Apical has zymogen granules, rER occupies basal
Light Microscopy resolution and formula
200nm, d = 0.61lambda/NA
TEM
Thin section, freeze fractured, Dehydrated and embedded in resin, negative staining in heavy metal salt solution, contrast visualized by EM grid
SEM
Sample dehydrated, coat in gold, electron beam used to scan and determine 3D structure
How does Coomassie work
Binds to protein, and dye becomes blue in colour. Absorption peak when bound to protein is 595nm, which corresponds to amount of protein
Example of Charged aa
Arginine, Histidine - positive
Glutamic and Aspartic acid - positive
Polar and Non-polar aa
Polar: Serine, Glutamine, Asparagine
Non-polar: Alanine, Phenylalanine, Leucine, Isoleucine
How Lysozymes work
Hydrolyze glycosidic bond between NAM and NAG, only work on Gram-positive bacteria
Denaturing/Refolding of protein
SDS: Unfolds protein
DTT: Breaks disulphide bridges
Oxidation: Re-forms S-S bridges (protein needs to refold first for this to occur correctly)
Photosynthesis e acceptors, and respiration inhibitors
Ferricyanide: Accepts e- from both PSs
Phenyl Quinone: Only accepts from PSII
KCN added to inhibit Cytochrome c oxidase (respiration cannot occur)
How to tell if LOF is same gene or different gene
Form diploid by mating two different strains on non-selective plate, allow diploid to grow
Transfer diploid to selection plate - if same gene, then will not grow and vice versa (complementation test)
How to tell if phenotype comes from the same gene
Cross with WT on plate with complete medium, obtain diploid. Transfer diploid to minimal medium plate, starve C and N induces meiosis and spore formation.
Plate spores on non-selective plate, allowing to grow. Transfer the spores to selection plate, and confirm single-factor ratio = 1
Use of Ca2+ in transformation
Ca2+ overcomes electrostatic repulsion between -vely charged plasmid DNA and LPS in bacterial cell wall. Increases likelihood of uptake of plasmid by bacteria.
