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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
Q

what is TAE?

A

tris (pH), acetate (salts), EDTA (removes impurities)

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26
Q

what is sybrsape?

A

fluorescent molecule that binds cDNA and causes the bands to glow

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27
Q

what is the process called that allows the bands to glow during PCR? what is the energy source for this?

A

fluorescence
- UV light

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28
Q

in the PCR gel, which pieces move the furthest and quickest? which charge do they move to?

A

smaller pieces
- move toward positive end (red)

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29
Q

what is the operculum?

A

flap under chin

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30
Q

what is a blastema?

A

injury regrowth (mitosis)

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31
Q

what is the apical epithelial cap?

A

covering of the blastema
- sends Fgf signals to nearby cells

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32
Q

what is the regenerated limb called when it starts to flatten into a paddle shape?

A

pallette

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33
Q

what cell type produces the new tissue of a blastema?

A

mesenchyme

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34
Q

what are the five requirements of regeneration?

A
  • wound epithelium (skin)
  • dedifferentiated progenitor cells
  • blastema cell proliferation
  • sufficient nerve supply
  • positional & patterning info
35
Q

what is the anesthetic we used?

A

MS-222 0.3%

36
Q

what is the fixation solution we used? what does it do?

A

4% paraformaldehyde
- prevents decay

37
Q

what is the cryoprotectant solution we used? what does it do?

A

30% sucrose
- sucks water out of cells and prevent ice formation

38
Q

what type of chromosomes do flies have?

A

polytene (4)
- makes it easy to visible see mutations on chromosomes

39
Q

what is different about a the appearance of a male fly compared to a female?

A

smaller, black, have sex limbs

40
Q

what is the staining process we used for monoclonal antibodies? what is the function?

A

immunohistochemistry
- allows Abs to bind antigens in a a specific manner
- gives location and identifies specific cells

41
Q

what is the difference between monoclonal Ab vs polyclonal Ab?

A

monoclonal
- more specific
- binds to only one epitope / protein

polyclonal
- non-specific
- binds to many epitopes / proteins

42
Q

what is the fab region?

A

region that binds to the protein

43
Q

what is the fc region?

A

epitope on the primary Ab for the secondary Ab to bind to
- specific to the animal
- INDIRECT METHOD

44
Q

what type of cells do monoclonal antibodies come from?

A

spleen cells (B-cells)

45
Q

describe the process of making monoclonal antibodies

A
  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
Q

what are the three things in the HAT medium?

A

hypoxanthine
aminopterin
thymidine

47
Q

what does hypoxanthine do? which pathway is it active in?

A

converted to G nucleotide
- salvage pathway

48
Q

what does aminopterin do? which pathway is it active in?

A

blocks normal pathway to allow for salvage pathway to make G & T nucleotides
- main pathway

49
Q

what does thymidine do? which pathway is it active in?

A

makes the T nucleotide
- salvage pathway

50
Q

what does HGPRT do? which pathway is it active in?

A

turns hypoxanthine into G nucleotide
- salvage pathway

51
Q

How does the HAT medium kill -/- cells but allows fused antibodies to survive?

A

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
Q

what does a peltier do?

A

in the cryostat that sucks the heat out

53
Q

what does OCT compound do?

A

freezes at same consistency as tissue to allow for cross sectioning

54
Q

what was the micron selection we used in the cryostat?

A

20 microns

55
Q

what was the dilution of the primary antibody?

A

1:10

56
Q

what was the secondary antibody we used?

A

goat antimouse IgG AlexaFluor488nm conjugated
- made by goat
- binds to mice DNA
- 488 is the color that is absorbed

57
Q

what does the secondary antibody do?

A

binds to primary antibody and glows GREEN
- functions as a tag for the gene of interest

58
Q

what does wheat germ agglutinin do? what dilution did we use?

A

binds to cell MB and glows RED
- 1:100
- apart of counterstain mix
- 594nm

59
Q

what was the dilution of the secondary antibody?

A

1:200

60
Q

what does 5% normal goat serum do?

A

contains non-specific Ab that binds to the sticky areas
- apart of blocking solution

61
Q

what does 0.3% triton / tween do?

A

soap that gets into cell MB and breaks it apart
- apart of blocking solution

62
Q

what is PBS?

A

phosphate buffer saline
- same osmolarity as cells
- washes away leftover liquid

63
Q

what does ProLong antifade with DAPI do?

A

binds to DNA and glows BLUE (406nm)
- prevents refraction of light

64
Q

what does Epi Fluorescence do?

A

absorbs e-, they get excited, fall back to OG state & release orb of light
- views multiple planes
- uses LED as a light source

65
Q

describe the difference between absorbed light and emitted light?

A

absorbed = short wavelength, high energy
emitted = long wavelength, low energy

66
Q

which way is higher energy shifted?

A

LEFT

67
Q

what is the range of visible light?

A

400-700nm

68
Q

what is the difference between a confocal microscope and the Epi fluorescence?

A

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
Q

what does the confocal pinhole do?

A

light we want is filtered through by blocking the unfocused light rays

70
Q

what do the dichroic mirrors do?

A

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
Q

what does the photomultipler tube do?

A

captures photons & produces a stream of electrical charge

72
Q

what is optical sectioning? what makes this feature in the confocal different compared to epi fluorescence?

A

optical sectioning is looking at a single layer / plane
- in epi, you are viewing all planes / layers

73
Q

what is a homogenizer?

A

makes tissue small enough for the buffer to enter and dissolve cell MBs

74
Q

what is the function of Hensen’s Node?

A

part of dorsal lip in gastrulation that helps establish anterior-posterior axis

75
Q

what are five types of RNA?

A

mRNA, tRNA, miRNA, lnRNA, rRNA, snRNA, hnRNA

76
Q

what does elution solution do?

A

gets RNA off filter to dissolve back into solution

77
Q

what is resolution (resolving power)?

A

distance
- distance between points that you can visibly see

78
Q

what is magnification?

A

size

79
Q

what is contrast?

A

color

80
Q

what is the equation for resolving power?

A

RP = wavelength / 2 x numerical aperture
- RP should be a small number
- NA to be big & wavelength to be small

81
Q

what are 5 things needed to turn mRNA into cDNA?

A
  1. verso enzyme (RNA dep, DNA poly)
  2. cDNA synthesis buffer
  3. anchored oligo-dT (primer)
  4. dNTP mix
  5. RT enhancer
82
Q

what are five things needed for PCR?

A
  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
Q

what does the rhombencephalon become?

A

nervous system

84
Q

what are the five parts of the brain we need to know for chick anatomy in order, starting with the front of the head?

A
  1. telencephalon (T)
  2. diencephalon (D)
  3. mesencephalon (MS)
  4. metencephalon (MT)
  5. myelencephalon (MY)

Developmental Biology (17 decks)

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