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Developmental Biology > Lab FInal Exam > Flashcards

Lab FInal Exam Flashcards

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1
Q

what is the scientific name of the axolotl we studied?

A

Ambystoma Mexicanum

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2
Q

what is the name of the guy we get our axolotl’s from?

A

Randal Voss

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3
Q

what is the cloaca?

A

opening for excretion and reproduction in axolotls

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4
Q

how do axolotls reproduce?

A

internal fertilization
- male lays a group of sperm (spermatophore) in a jelly coat on the ground
- female comes by and sits on the sperm and sucks up sperm through her cloaca

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5
Q

what does neotony mean?

A

retains juvenile form, no metamorphosis

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6
Q

what does a lysis buffer do?

A

stops RNases from destorying the RNA

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7
Q

what is cDNA?

A

RNA reverse transcribed

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8
Q

what is the enzyme that reads the mRNA and makes cDNA?

A

Reverse Transcriptase enzyme (RNA-dependent DNA polymerase)

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9
Q

what is the enzyme that is used to read the cDNA during PCR?

A

Taq Polymerase
- DNA-dependent DNA polymerase

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10
Q

what does the verso enzyme do?

A

polymerase that makes the long cDNA in PCR

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11
Q

what does the cDNA synthesis buffer do?

A

contains salts & ions to ensure its at the right pH

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12
Q

what does the anchored oligo-dT do?

A

binds to the poly-A-tail (TTTTT) and makes a primer

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13
Q

what does the dNTP mix do?

A

adds bases to make the cDNA during PCR

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14
Q

what does the RT enhancer do?

A

removes contaminations to ensure what is left is cDNA

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15
Q

what is the 5’ forward primer we used? (upstream)

A

G-U26

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16
Q

what is the 3’ reverse primer we used? (downstream)

A

G-L64

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17
Q

what type of growth does PCR do?

A

exponential growth

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18
Q

what are the three temperatures and functions of the heat block used prior to PCR?

A

50 (makes cDNA)
95 (stops removal of double stranded DNA, inactivates enhancer)
4

*takes 50 minutes

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19
Q

what are the three temperatures and functions of PCR amplification?

A

95 (denature)
62 (anneal)
72 (elongation)

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20
Q

what is a housekeeping gene? which one did we use?

A

gene found in all cells
- GAPDH

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21
Q

what are the four types of PCR? describe them

A

endpoint = check if the gene is in the genome & if the primers work

reverse transcription = RNA -> cDNA, makes a template

semiquantitative = eyeball to see how much total produce was made

quantitative = fluorescent primers that glow when its double stranded, precise measurement

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22
Q

what type of PCR did we do?

A

RT-sq

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23
Q

what is differential adhesion?

A

the theory that three germ layers organize themselves in patterns due to specific affinities and cadherins
- hierarchy of cell types sorting themselves out
- higher surface tension tissue in the middle

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24
Q

who created the differential adhesion theory?

A

Malcolm Steinberg

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25
what is TAE?
tris (pH), acetate (salts), EDTA (removes impurities)
26
what is sybrsape?
fluorescent molecule that binds cDNA and causes the bands to glow
27
what is the process called that allows the bands to glow during PCR? what is the energy source for this?
fluorescence - UV light
28
in the PCR gel, which pieces move the furthest and quickest? which charge do they move to?
smaller pieces - move toward positive end (red)
29
what is the operculum?
flap under chin
30
what is a blastema?
injury regrowth (mitosis)
31
what is the apical epithelial cap?
covering of the blastema - sends Fgf signals to nearby cells
32
what is the regenerated limb called when it starts to flatten into a paddle shape?
pallette
33
what cell type produces the new tissue of a blastema?
mesenchyme
34
what are the five requirements of regeneration?
- wound epithelium (skin) - dedifferentiated progenitor cells - blastema cell proliferation - sufficient nerve supply - positional & patterning info
35
what is the anesthetic we used?
MS-222 0.3%
36
what is the fixation solution we used? what does it do?
4% paraformaldehyde - prevents decay
37
what is the cryoprotectant solution we used? what does it do?
30% sucrose - sucks water out of cells and prevent ice formation
38
what type of chromosomes do flies have?
polytene (4) - makes it easy to visible see mutations on chromosomes
39
what is different about a the appearance of a male fly compared to a female?
smaller, black, have sex limbs
40
what is the staining process we used for monoclonal antibodies? what is the function?
immunohistochemistry - allows Abs to bind antigens in a a specific manner - gives location and identifies specific cells
41
what is the difference between monoclonal Ab vs polyclonal Ab?
monoclonal - more specific - binds to only one epitope / protein polyclonal - non-specific - binds to many epitopes / proteins
42
what is the fab region?
region that binds to the protein
43
what is the fc region?
epitope on the primary Ab for the secondary Ab to bind to - specific to the animal - INDIRECT METHOD
44
what type of cells do monoclonal antibodies come from?
spleen cells (B-cells)
45
describe the process of making monoclonal antibodies
1. antigen of interest is injected into a mouse 2. B-cells are made in spleen and collected (+/+ HGPRT) 3. B-cells (+/+) and cancer cells (-/-) are put together 4. polyethylene glycol (PEG) is added to fuse MB's and nuclei together 5. cells are grown in a HAT medium 6. salvage pathway makes the DNA replicate 7. only hybridoma cells survive (Bcells+cancer) 8. hybridoma cells make mAb
46
what are the three things in the HAT medium?
hypoxanthine aminopterin thymidine
47
what does hypoxanthine do? which pathway is it active in?
converted to G nucleotide - salvage pathway
48
what does aminopterin do? which pathway is it active in?
blocks normal pathway to allow for salvage pathway to make G & T nucleotides - main pathway
49
what does thymidine do? which pathway is it active in?
makes the T nucleotide - salvage pathway
50
what does HGPRT do? which pathway is it active in?
turns hypoxanthine into G nucleotide - salvage pathway
51
How does the HAT medium kill -/- cells but allows fused antibodies to survive?
Cancer cells do not have HGPRT(-/-) which is needed in the salvage pathway to produce new nucleotides, but they are immortal. B-cells have HGPRT (+/+),but since they aren't immortal, they will not survive. The hybridoma cells are a fusion of both cancer and B-cells. Since the hybridoma cells have the characteristics of being immortal and having the enzyme HGPRT, these are the only cells that will survive.
52
what does a peltier do?
in the cryostat that sucks the heat out
53
what does OCT compound do?
freezes at same consistency as tissue to allow for cross sectioning
54
what was the micron selection we used in the cryostat?
20 microns
55
what was the dilution of the primary antibody?
1:10
56
what was the secondary antibody we used?
goat antimouse IgG AlexaFluor488nm conjugated - made by goat - binds to mice DNA - 488 is the color that is absorbed
57
what does the secondary antibody do?
binds to primary antibody and glows GREEN - functions as a tag for the gene of interest
58
what does wheat germ agglutinin do? what dilution did we use?
binds to cell MB and glows RED - 1:100 - apart of counterstain mix - 594nm
59
what was the dilution of the secondary antibody?
1:200
60
what does 5% normal goat serum do?
contains non-specific Ab that binds to the sticky areas - apart of blocking solution
61
what does 0.3% triton / tween do?
soap that gets into cell MB and breaks it apart - apart of blocking solution
62
what is PBS?
phosphate buffer saline - same osmolarity as cells - washes away leftover liquid
63
what does ProLong antifade with DAPI do?
binds to DNA and glows BLUE (406nm) - prevents refraction of light
64
what does Epi Fluorescence do?
absorbs e-, they get excited, fall back to OG state & release orb of light - views multiple planes - uses LED as a light source
65
describe the difference between absorbed light and emitted light?
absorbed = short wavelength, high energy emitted = long wavelength, low energy
66
which way is higher energy shifted?
LEFT
67
what is the range of visible light?
400-700nm
68
what is the difference between a confocal microscope and the Epi fluorescence?
confocal uses a software that combines images to make a 3D images by using lasers as a source of light - views a single plane
69
what does the confocal pinhole do?
light we want is filtered through by blocking the unfocused light rays
70
what do the dichroic mirrors do?
rotate to focus lasers on the slide - allows high energy light of a certain wavelength to pass through, while light of other wavelengths is reflected
71
what does the photomultipler tube do?
captures photons & produces a stream of electrical charge
72
what is optical sectioning? what makes this feature in the confocal different compared to epi fluorescence?
optical sectioning is looking at a single layer / plane - in epi, you are viewing all planes / layers
73
what is a homogenizer?
makes tissue small enough for the buffer to enter and dissolve cell MBs
74
what is the function of Hensen's Node?
part of dorsal lip in gastrulation that helps establish anterior-posterior axis
75
what are five types of RNA?
mRNA, tRNA, miRNA, lnRNA, rRNA, snRNA, hnRNA
76
what does elution solution do?
gets RNA off filter to dissolve back into solution
77
what is resolution (resolving power)?
distance - distance between points that you can visibly see
78
what is magnification?
size
79
what is contrast?
color
80
what is the equation for resolving power?
RP = wavelength / 2 x numerical aperture - RP should be a small number - NA to be big & wavelength to be small
81
what are 5 things needed to turn mRNA into cDNA?
1. verso enzyme (RNA dep, DNA poly) 2. cDNA synthesis buffer 3. anchored oligo-dT (primer) 4. dNTP mix 5. RT enhancer
82
what are five things needed for PCR?
1. template DNA 2. taq poly (DNA dep, DNA poly) 3. reaction buffer 4. dNTP mix 5. upstream & downstream primers (G-U26, G-L64)
83
what does the rhombencephalon become?
nervous system
84
what are the five parts of the brain we need to know for chick anatomy in order, starting with the front of the head?
1. telencephalon (T) 2. diencephalon (D) 3. mesencephalon (MS) 4. metencephalon (MT) 5. myelencephalon (MY)

Developmental Biology (17 decks)

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