Term
| __: gathers the diffuse rays from light source and illuminates speciman |
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Definition
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Term
| __: collects light rays previously focused on speciman |
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Definition
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Term
| __: uses real enlarged image from objective lens and forms from it an enlarged virtual image |
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Definition
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Term
| __: the smallest unit/distance at which 2 objects can be seen as seperate from each other |
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Definition
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Term
| resolution = resolving power (d) = ___ |
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Definition
| .61 lambda / n sin alpha (NA of objective lens) |
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Term
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Definition
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Term
| when the sieve is rinsed with h2o, the embryos will aggregate together and flow to the center of the sieve, this means that the __ has a __ character |
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Definition
| vitelline membrane; non-polar |
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Term
| the embryos are then transferred to the tube containing the __ mix, from step 1, this step __ surrounding the embryos |
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Definition
| heptane-MeOH mix; dissolves the waxy coat (vitelline membrane) |
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Term
| Place the tube of embryos in the mix in an eppendorf rack, the heptane and MeOH will seperate into distinct layers. the upper layer will be __, and lower will be __. only the embryos with with a __ will sink to the bottom of the tube. |
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Definition
| heptane; MeOH; dissolved vitelline membrane |
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Term
| once this is done, use a pasteur pippete to remove the __ layer |
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Definition
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Term
| next, fill the tube with __ and allow the embryos to incubate for an hour to permanently fix their __ |
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Definition
| MeOH; internal structures |
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Term
| how would you compare the 4x and 10x objectives in terms of differences in magnification and resolution (can you see the perimeter of the nucleus with both objectives or only with one?) |
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Definition
| could not see perimeter of nucleus with 4x, but could with 10x, picture became more clear with 10x |
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Term
| how would you compare the 40x and 10x objectives in terms of differences in magnification and resolution (can you see the perimeter of the nucleus with both objectives or only with one?) |
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Definition
| could see perimeter of nucleus with both 10x and 40x, nucleus was much larger with 40x but harder to find |
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Term
| use each 4x, 10x, and 40x objectives to view the unstained onion epidermis. which is the lowest power objective needed to clearly see the nucleus and its perimeter in this specimen. |
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Definition
| 40x, you can kind of see the nucleus with 10x, but to clearly see the nucleus you need 40x |
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Term
| make adjustments to the condenser iris opening to achieve optimal clarity. how does this adjustment compare to the one that gives the best clarity of the stained specimen? |
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Definition
| the stained specimen requires a little bit more light than the unstained specimen |
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Term
| when this is repeated with the slides of the stained and unstained squamous cheek cells. which is the lowest power objective required to see the nucleus and resolve its perimeter in this stained specimen. |
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Definition
| the nuclei can be seen somewhat with 4x but can clearly be made out with 10x |
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Term
how do adjustments to the condensor iris affect the visibility of each specimen?
- changes in light affect __ and __ |
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Definition
as before, the stained needs a bit more light than the unstained, but to much light at all will destroy visibility.
- contrast; resolution |
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Term
| the lenses with greater na decrease __ which increase __ |
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Definition
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Term
| what color will a dapi stained molecule appear? |
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Definition
| blue (absorbs at 358nm, emits at 461nm) |
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Term
| what color will a fluorescein labeled molecule appear |
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Definition
| green (absorbs at 494nm, emits at 521nm) |
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Term
| what color will a texas red labeled molecule appear? |
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Definition
| greenish-red / yellow (absorbs at 589nm, emits at 615nm) |
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Term
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Definition
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Term
methods for labeling molecules wit fluorescent tags
1) direct attachment through __ binding of fluorescent stain. this type of labeling is done through fluorescent molecules that display natural binding affinity for a molecule of interest. although this method is less universally useful, it is used routinely to label __ through __ which stains __ |
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Definition
| non-covalent; dna; dapi staining; blue |
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Term
| 2) immunofluorescence: indirect attachment through a fluorescently labeled __. this commonly used method uses an __ to target a fluorescent tag to a protein of interest. cell biologists make use of antibody specificity as a means for targeting a fluorescent tag to the protein it recognizes. the fluorescent tag can either be __ attached to the antibody or it can by __ attached through a fluorescently tagged ___ that recognizes that antibody. The __ method is more commonly used. |
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Definition
| antibody; antibody; directly; indirectly; secondary antibody; indirect |
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Term
| 3) direct attachment through __ linkage. although it requires more work, it is also possible to chemically link a fluorescent tag to any protein of interest. fluorescent molecules with chemically reactive side chains are made commercially available for catalyzing the covalent linkage of a fluorescent tag to a purified protein in a __ reaction. the __ labeled protein must then be injected into a living cell, in which it can be monitered by fluorescence microscopy. |
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Definition
| covalent linkage; test tube; in vitro |
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Term
| 4) in vivo expression of __. this method allows the behavior of a labeled protein to be monitored in a __. this method requires the cell to produce a __, which is transferred into the genome of a living organism, where it is transcribed and translated into a gfp tagged version of the protein of interest. |
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Definition
| gfp fusion proteins; living cell; recombinant gene |
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Term
| methods 1 and 2 requires that the cells be treated with a __ before __. methods 3 and 4 require a __ |
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Definition
| fixative; staining; living specimen |
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Term
| how does the anti-histone immunostaining pattern (method #2) compare to the dapi staining pattern (method #1)? |
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Definition
| the anti-histone stains the protein (histones) around the dna, while the dapi stains the dna directly. however, both of theser are in the nucleus so the images appear similar |
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Term
| the dapi only allow you to see the __; immunostaining allows you to see the __ or __ |
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Definition
| nucleus; histones; the neurons around the nucleus |
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Term
| why might a researcher choose method 3 or 4 over method 1 or 2? |
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Definition
| methods 3 or 4 require a live specimen, methods 1 and 2 require a dead speciman |
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Term
| can you think of any potential technical problems that might arise with gfp tagging? |
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Definition
| it could change the structure/conformation of the protein |
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Term
| what specialized reagents would need to be made for method #2? |
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Definition
| you need the antibodies for a particular protein |
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Term
| when might a researcher choose method 1 over method 2 |
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Definition
| it is more straight forward and easier, mainly used to view dna in the nucleus, so to view the nucleus |
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Term
| what advantage did each of these methods give over the iodine staining used to observe the squamos cheek cells in lab 1a? |
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Definition
| the iodine staining just increased the magnification, and allowed for increased magnification. methods 1 and 2 are more specific and allow for increased scrutiny of certain areas. methods 3 and 4 allow viewrs to observe parts of the cell in action. |
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Term
| what specialized reagent would need to be made before you could do live imaging of gfp tagged protein? |
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Definition
| you need to insert the recombinent gene and allow it to trascribe and translate the gfp tagged protein |
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Term
| a first round of low speed centrifugation will divide the mixture into __ and __ |
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Definition
| low speed pellet (large organelles such as nucleus); low speed supernatant fraction (all other) |
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Term
| the LSS fraction is further subjected to high speed centrifugation which divides into __ and __ |
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Definition
| high speed pellet (mitochondria); high speed supernatant (golgi and ribosome) |
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Term
| __ stains dna while __ stains mitochondria |
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Definition
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Term
| orecin staining would be expected in the __ a lot, and maybe a little in the __ |
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Definition
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Term
| janus green staining would be expected in the __ and __ |
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Definition
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Term
| what is the purpose of the assasys done in the absence of succinate? what result do you expect to observe in these reactions |
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Definition
| the reaction lacking succinate is a negative control for the assay. |
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Term
| __ is an enzyme of the tca cycle that is an integral component of the inner mitochondrial membrane. __ acts as an artificial electron acceptor |
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Definition
| succinate dehydrogenase; dcip |
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Term
| the goal in lab 3 is to determine the effect of competitor which is ___ |
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Definition
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Term
-noncompetitive: reduces __, does not affect __
-competitive: increases __, does not affect __ |
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Definition
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Term
| in a lineweaver burk plot, both the competitive and noncompetitive will have increased __ |
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Definition
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Term
| michaelis menten equation: |
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Definition
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Term
lineweaver burk plot formula:
- y intercept:
- x intercept: |
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Definition
(1/Vo) = (Km/Vmax)(1/S) + (1/Vmax)
- 1/Vmax
- -1/Km |
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