UIC BIOS 223 - Micropipettor use and the Bradford Assay

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Micropipettor use and the Bradford AssayKnow what volume range P20, P200, P1000 are used forKnow how you would make 1:10, 1:100 dilutions of a sampleWhat was the purpose of the Bradford assay?What color is the solution you are observing?What are the four main steps of Bradford assay (in background)?Termite SymbiontsKnow the general background of termitesLook at the images of what you expect to see after you stain?What staining materials did you use and the purpose?What part of the termite body did you study?Difference between live and stained sampleWhat was the magnification used to observe?Fluorescent dyes, DrosophilaList the stains used in the lab, what do they stain and the color?Which stage of Drosophila was used for this study? Which specific organ?What is a polytene chromosome? Where are they found?Know the general procedure of how you stained for each stainSaccharomyces and endocytosisKnow the growth pattern of yeast. What time point is lag, log and stationary phase?What is the relevance of this lab?The purpose of this lab is to observe endocytosisWhat dye is used and how does it work?What color should you see under the microscope and where?Which microscope fluorescence filter(s) was used? What magnification?What OD was used?If you were to perform a spectrophotometry, what steps will you follow?SDS-PAGE, Protein extractionKnow how to create a cladogramWhat is SDS-PAGE used for? Know the general stuffWhat is the stain used to see bands on the gel?How long and volts was the gel run (according to protocol)?Why was the Bradford assay performed?What’s the purpose of the ladder?What is the purpose of the overall experiment?Which fish species were used?Yeast Mating ResponseWhat are the two mating types?Know the haploid and diploid mating statesWhat is the chemical secreted for signaling/sensing?PheromonesRead of the background, overall signaling pathway.Fractionation by Centrifugation, MitochondriaWhat is the overall purpose of this technique?What speeds were used? What was in the pellet and supernatant for each speed?Know the general backgroundYeast AutophagyWhich dye was used to observe autophagy?What are the different organelles created (look at the quiz questions)?Which stains were used and what did they stain? DAPI – DNA FM464 – lipid membranesGFP – ATG8 ProteinsWhich filters were used?Look at the class data for images and review the background.Deciliation, Cilia growth, Cell fixationRead the background and procedure for both parts.What is the purpose and overall procedure?What is the purpose of immunofluorescence?What were the observational results over time?The cells overtime were able to move more and moreMicroscopically, what is the most obvious feature of Tetrahymena?What is the function of Dibucaine in this experiment?Micropipettor use and the Bradford AssayKnow what volume range P20, P200, P1000 are used forP20: volumes up to 20uL, P200: volumes up to 200uL, P1000: volumes uo to 1000uL or 1mLKnow how you would make 1:10, 1:100 dilutions of a sampleDivide volume by total dilutionWhat 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?BlueWhat 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 SymbiontsKnow the general background of termites- Internal organs (viscera) contains symbionts under observation- Symbionts help termite digest the cellulose in wood fibers the termites ingestLook at the images of what you expect to see after you stain?large and covered in ciliaWhat staining materials did you use and the purpose?- Bouin’s fixative: fixed sample onto slide. Stained sample yellow.- Ethanol: washed yellow pigment from sample1- Hematoxylin/FeCl2/I2: o Hematoxylin: purple staino FeCl2: binds/fixes stain and sample. Also used to remove excessstain.o I2: oxidizes hematoxylin to obtain Hematein dyeWhat part of the termite body did you study?Viscera, internal organs in the abdomenDifference between live and stained sample- Live: no stain harder to observe symbionts because cilia not fully visible- Stained: purple cilia visibleWhat was the magnification used to observe?Between staining steps 10X, after stained 40X and 100XFluorescent dyes, DrosophilaList the stains used in the lab, what do they stain and the color?- Aceto-orcein: binds to chromosomes within the nucleuso Pinkish red color, best seen under 100X with bright field Clumps in salivary gland- Arcidine orange: stains DNA o Green Also binds to other structures within the cell- DAPI: binds to DNA in the nucleuso Blue Which stage of Drosophila was used for this study? Which specific organ?Larva, salivary glandWhat 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: 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.- Arcidine orange: 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- DAPIo Add drop of DAPI onto slide, add gland, allow to stain for 5 mins, then add drop of phosphate bufferSaccharomyces and endocytosisKnow 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 double2- 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 endocytosisWhat is endocytosis?It is the internalization of degraded parts of the cell membrane for renewal or recyclingWhat 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


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UIC BIOS 223 - Micropipettor use and the Bradford Assay

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