Lab FInal Exam Flashcards
what is the scientific name of the axolotl we studied?
Ambystoma Mexicanum
what is the name of the guy we get our axolotl’s from?
Randal Voss
what is the cloaca?
opening for excretion and reproduction in axolotls
how do axolotls reproduce?
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
what does neotony mean?
retains juvenile form, no metamorphosis
what does a lysis buffer do?
stops RNases from destorying the RNA
what is cDNA?
RNA reverse transcribed
what is the enzyme that reads the mRNA and makes cDNA?
Reverse Transcriptase enzyme (RNA-dependent DNA polymerase)
what is the enzyme that is used to read the cDNA during PCR?
Taq Polymerase
- DNA-dependent DNA polymerase
what does the verso enzyme do?
polymerase that makes the long cDNA in PCR
what does the cDNA synthesis buffer do?
contains salts & ions to ensure its at the right pH
what does the anchored oligo-dT do?
binds to the poly-A-tail (TTTTT) and makes a primer
what does the dNTP mix do?
adds bases to make the cDNA during PCR
what does the RT enhancer do?
removes contaminations to ensure what is left is cDNA
what is the 5’ forward primer we used? (upstream)
G-U26
what is the 3’ reverse primer we used? (downstream)
G-L64
what type of growth does PCR do?
exponential growth
what are the three temperatures and functions of the heat block used prior to PCR?
50 (makes cDNA)
95 (stops removal of double stranded DNA, inactivates enhancer)
4
*takes 50 minutes
what are the three temperatures and functions of PCR amplification?
95 (denature)
62 (anneal)
72 (elongation)
what is a housekeeping gene? which one did we use?
gene found in all cells
- GAPDH
what are the four types of PCR? describe them
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
what type of PCR did we do?
RT-sq
what is differential adhesion?
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
who created the differential adhesion theory?
Malcolm Steinberg
what is TAE?
tris (pH), acetate (salts), EDTA (removes impurities)
what is sybrsape?
fluorescent molecule that binds cDNA and causes the bands to glow
what is the process called that allows the bands to glow during PCR? what is the energy source for this?
fluorescence
- UV light
in the PCR gel, which pieces move the furthest and quickest? which charge do they move to?
smaller pieces
- move toward positive end (red)
what is the operculum?
flap under chin
what is a blastema?
injury regrowth (mitosis)
what is the apical epithelial cap?
covering of the blastema
- sends Fgf signals to nearby cells
what is the regenerated limb called when it starts to flatten into a paddle shape?
pallette
what cell type produces the new tissue of a blastema?
mesenchyme
what are the five requirements of regeneration?
- wound epithelium (skin)
- dedifferentiated progenitor cells
- blastema cell proliferation
- sufficient nerve supply
- positional & patterning info
what is the anesthetic we used?
MS-222 0.3%
what is the fixation solution we used? what does it do?
4% paraformaldehyde
- prevents decay
what is the cryoprotectant solution we used? what does it do?
30% sucrose
- sucks water out of cells and prevent ice formation
what type of chromosomes do flies have?
polytene (4)
- makes it easy to visible see mutations on chromosomes
what is different about a the appearance of a male fly compared to a female?
smaller, black, have sex limbs
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
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
what is the fab region?
region that binds to the protein
what is the fc region?
epitope on the primary Ab for the secondary Ab to bind to
- specific to the animal
- INDIRECT METHOD
what type of cells do monoclonal antibodies come from?
spleen cells (B-cells)
describe the process of making monoclonal antibodies
- antigen of interest is injected into a mouse
- B-cells are made in spleen and collected (+/+ HGPRT)
- B-cells (+/+) and cancer cells (-/-) are put together
- polyethylene glycol (PEG) is added to fuse MB’s and nuclei together
- cells are grown in a HAT medium
- salvage pathway makes the DNA replicate
- only hybridoma cells survive (Bcells+cancer)
- hybridoma cells make mAb
what are the three things in the HAT medium?
hypoxanthine
aminopterin
thymidine
what does hypoxanthine do? which pathway is it active in?
converted to G nucleotide
- salvage pathway
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
what does thymidine do? which pathway is it active in?
makes the T nucleotide
- salvage pathway
what does HGPRT do? which pathway is it active in?
turns hypoxanthine into G nucleotide
- salvage pathway
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.
what does a peltier do?
in the cryostat that sucks the heat out
what does OCT compound do?
freezes at same consistency as tissue to allow for cross sectioning
what was the micron selection we used in the cryostat?
20 microns
what was the dilution of the primary antibody?
1:10
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
what does the secondary antibody do?
binds to primary antibody and glows GREEN
- functions as a tag for the gene of interest
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
what was the dilution of the secondary antibody?
1:200
what does 5% normal goat serum do?
contains non-specific Ab that binds to the sticky areas
- apart of blocking solution
what does 0.3% triton / tween do?
soap that gets into cell MB and breaks it apart
- apart of blocking solution
what is PBS?
phosphate buffer saline
- same osmolarity as cells
- washes away leftover liquid
what does ProLong antifade with DAPI do?
binds to DNA and glows BLUE (406nm)
- prevents refraction of light
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
describe the difference between absorbed light and emitted light?
absorbed = short wavelength, high energy
emitted = long wavelength, low energy
which way is higher energy shifted?
LEFT
what is the range of visible light?
400-700nm
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
what does the confocal pinhole do?
light we want is filtered through by blocking the unfocused light rays
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
what does the photomultipler tube do?
captures photons & produces a stream of electrical charge
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
what is a homogenizer?
makes tissue small enough for the buffer to enter and dissolve cell MBs
what is the function of Hensen’s Node?
part of dorsal lip in gastrulation that helps establish anterior-posterior axis
what are five types of RNA?
mRNA, tRNA, miRNA, lnRNA, rRNA, snRNA, hnRNA
what does elution solution do?
gets RNA off filter to dissolve back into solution
what is resolution (resolving power)?
distance
- distance between points that you can visibly see
what is magnification?
size
what is contrast?
color
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
what are 5 things needed to turn mRNA into cDNA?
- verso enzyme (RNA dep, DNA poly)
- cDNA synthesis buffer
- anchored oligo-dT (primer)
- dNTP mix
- RT enhancer
what are five things needed for PCR?
- template DNA
- taq poly (DNA dep, DNA poly)
- reaction buffer
- dNTP mix
- upstream & downstream primers (G-U26, G-L64)
what does the rhombencephalon become?
nervous system
what are the five parts of the brain we need to know for chick anatomy in order, starting with the front of the head?
- telencephalon (T)
- diencephalon (D)
- mesencephalon (MS)
- metencephalon (MT)
- myelencephalon (MY)
