Micropipettor use and the Bradford Assay Know what volume range P20 P200 P1000 are used for P20 volumes up to 20uL P200 volumes up to 200uL P1000 volumes uo to 1000uL or 1mL Know how you would make 1 10 1 100 dilutions of a sample Divide volume by total dilution What was the purpose of the Bradford assay Used to measure the amount of protein in a sample What color is the solution you are observing Blue What are the four main steps of Bradford assay in background Preparation of dilution or known protein standards and preparation of unknowns Add Bradford dye brown cationic form and incubate for 5 60 mins Observe binding by observing color change from brown to blue darker blue indicates high prtn conc then observe in spect At A595 Compile data into standard curve and determine unknown prtn conc Termite Symbionts Know the general background of termites Internal organs viscera contains symbionts under observation Symbionts help termite digest the cellulose in wood fibers the termites ingest Look at the images of what you expect to see after you stain large and covered in cilia What staining materials did you use and the purpose Bouin s fixative fixed sample onto slide Stained sample yellow Ethanol washed yellow pigment from sample 1 Hematoxylin FeCl2 I2 o Hematoxylin purple stain o FeCl2 binds fixes stain and sample Also used to remove excess stain o I2 oxidizes hematoxylin to obtain Hematein dye What part of the termite body did you study Viscera internal organs in the abdomen Difference between live and stained sample Live no stain harder to observe symbionts because cilia not fully visible Stained purple cilia visible What was the magnification used to observe Between staining steps 10X after stained 40X and 100X Fluorescent dyes Drosophila List the stains used in the lab what do they stain and the color Aceto orcein binds to chromosomes within the nucleus o Pinkish red color best seen under 100X with bright field Clumps in salivary gland Arcidine orange stains DNA Also binds to other structures within the cell DAPI binds to DNA in the nucleus o Green o Blue Which stage of Drosophila was used for this study Which specific organ Larva salivary gland What is a polytene chromosome Where are they found They are chromosomes that replicate normally but never separate They are found on the salivary glands or the larva Know the general procedure of how you stained for each stain Aceto orcein Arcidine orange DAPI o Drop stain onto slide place gland in stain allow to sit for 15 mins to stain add fresh stain then incubate for 2 mins o Drop acetic acid onto slide and place glands allow to sit for 15 mins then add drop of stain and incubate stain for 5 mins o Add drop of DAPI onto slide add gland allow to stain for 5 mins then add drop of phosphate buffer Saccharomyces and endocytosis Know the growth pattern of yeast What time point is lag log and stationary phase Lag phase slow emergence from dormancy Log phase rapid growth growth is double 2 Stationary phase stable growth often due to a growth limiting factor such as the depletion of an essential nutrient and or the formation of an inhibitory product such as an organic acid What is the relevance of this lab The purpose of this lab is to observe endocytosis What is endocytosis It is the internalization of degraded parts of the cell membrane for renewal or recycling What dye is used and how does it work FM 464 it binds to the lipids found on membranes liphophilic dye What color should you see under the microscope and where At time 0 the red dye should be seen on the outside of the cell and eventually it will be inside the cell Which microscope fluorescence filter s was used What magnification Rhodamine at 40X and 100X to view the dies DIC at 40X and 100X to see the cell normally What OD was used 600nm If you were to perform a spectrophotometry what steps will you follow Create cuvettes of proper dilution and a blank cuvette Blank machine prior to taking readings Place cuvettes in machine and record SDS PAGE Protein extraction Know how to create a cladogram What is SDS PAGE used for Know the general stuff What is the stain used to see bands on the gel How long and volts was the gel run according to protocol Coomasie Stain 150V for 1 hour Why was the Bradford assay performed The Bradford assay was used to find the correct amount of proteins that fell within the range of our standards which will be the correct amount in order for the protein that will move down the gel What s the purpose of the ladder The ladder was used as a guide to figure out the mass of the proteins Also it is used to ensure that the gel was run properly What is the purpose of the overall experiment The purpose of this experiment was to obtain a protein ladder gel and create a cladogram from it Which fish species were used 3 Yeast Mating Response What are the two mating types a and alpha Know the haploid and diploid mating states What is the chemical secreted for signaling sensing Pheromones Read of the background overall signaling pathway Fractionation by Centrifugation Mitochondria What is the overall purpose of this technique To separate different cell components What speeds were used What was in the pellet and supernatant for each speed 3 500 rpm supernatant growth media and other dense non cellular 2 300 rpm supernatant cell components pellet remaining glass beads and components cell debris 6 000 rpm pellet nuclei 10 000 rpm pellet mitochondria Which enzyme activity were you looking for Where is it found How does it work in terms of what you see in spectrophotometer What color change do you see in spectrophotometer Reduction of FAD in the presence of succinate dehydrogenase only occurred in the mitochondria Media should eventually lighten in color Know the general background Yeast Autophagy Which dye was used to observe autophagy FM4 64 dye What are the different organelles created look at the quiz questions Autophagosome autophagolysosome and autophagic bodies 4 Which stains were used and what did they stain DAPI DNA FM464 lipid membranes GFP ATG8 Proteins Which filters were used DIC DAPI FITC and Rhodamine Look at the class data for images and review the background Deciliation Cilia growth Cell fixation Read the background and procedure for both parts What is the purpose and overall procedure To observe cilia at different growth stages What is the purpose of immunofluorescence To observe the cilia lengths What were the observational results over time The cells