ch 8- microscopy and lab techniques Flashcards

1
Q

fixation

A

sticking cells to a slide

preserving them in their most lifelike state

chemical and heat fixation

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2
Q

what does staining do

A

often kills cells

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3
Q

electron microscopy

A

indirectly viewing cells through computer after bombarding them with electrons that pass through a magnetic field in a vaccuum

cells MUST be fixed and stained w metal coat and killed

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4
Q

stereo microscope

A

a dissection microscope

view surface of object

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5
Q

what can you see with a compound microscope

A

one cell thick live cells

have poor contrast without fixing and staining

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6
Q

phase contrast microscopy

A

viewing think samples with live cells

light refracted with annular ring creating phase shift
-can lead to halo effect

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7
Q

how to remove halo effect

A

phase plate or thinner samples

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8
Q

what is used in flourescence microscopy

A

dichroic filter
- allows certain wavelengths of light to be reflected and others to pass through

distortions or artifacts decrease resolution

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9
Q

confocal microscopy

A

visualizes flourescent objects

can be used without flouresence tagging

artifacts reduced by focusing beam of UV light on sample
-reduces intensity so samples must be illuminated longer

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10
Q

dark field micrsocopy

A

viewing unstained live cells bc the contrast is increased

only scattered light is viewed

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11
Q

scannning electron microscopy

A

high resolution 3D image of surface of DEHYDRATED sample

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12
Q

cryo SEM

A

sample frozen in liquid nitrogen instead of dehydrated

costly
produces artifacts

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13
Q

TEM

A

high resolution
2D images

internal structures

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14
Q

electron tomography

A

sandwiches TEM images to create 3D images of samples internal strutures

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15
Q

hemcytometer

A

counting chambers used to count cells

Gridded slide under microscope
-count cells in known area and extrapolate data

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16
Q

colony forming units (CFU)

A

estimate number of cells assuming one cell gives rise to one colony

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17
Q

automated cell counting

A

electrical resistance - counting cells by observing flow of elctricityf

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18
Q

flow cytometry

A

coutning cells by having them pass through a narrow tube and detecting them by a laser

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19
Q

lag phase

A

adaptation to prior cell division

line is horizontal, no increase in the number of bacteria

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20
Q

exponential (log) phase

A

rapid doubling

line has a positive slope

21
Q

stationary phase

A

occurs after the exponential log phase

line is horizontal

growth rate= death rate

22
Q

death phase

A

decline due to lack of food or other variable

negative slope

happens after stationary phase

23
Q

what does differential centrifugation separate

A

organelles and macromolecules based on size and density

24
Q

most dense to least dense organelles

A

nuclei
mitochondria/ chloroplasts
ER fragments
ribosomes

25
karyotyping
observing chromosomes under light microscope during METAPHASE used to diagnose conditions involving chromosomal abbherations, breakages, aneuploidies
26
how is DNA sequencing done
dideoxy chain termination/sanger sequencing next gen sequencing both may use shotgun sequencing
27
what is shotgun sequencing
cloned DNA genomes are cut into peices, sequenced, and recompiled to observe sequence overlaps used by sanger sequencing/ dideoxy chain termination and next gen sequencing
28
ddNTPs
dideoxynucleotides used in sanger sequencing lack two hydroxyl groups with with normal dNTPs for DNA pol to use - if they are added to a DNA strand, itll terminate elongation bc there is no 3' OH to form a new phosphodiester bond
29
single nucleotide polymorphisms
serve as markers for disease causing genes in humans
30
where do restriction enzymes cut DNA
at pallindromic sequences
31
RFLPs
restriction fragment length polymorphisms unique lengths of DNA from restriction enzymes; they allow for comparison between individuals by analyzing non-coding DNA (coding DNA is highly conserved).
32
exonucleases
result in sticky ends by cleaving nucleotides from the ends ofpolynucleotide chain
33
endonucleases
cleave within a polynucleotide chain result in either sticky or blunt ends
34
how is DNA fingerprinting done
looking at peoples RFLPs or short tandem repeats STR both unique stratches of DNA that allow for the identification of individuals
35
bacterial cloning
Cloning eukaryotic gene products in prokaryotic cells. Used to produce medicine. ● Protocol: Processed mRNA for eukaryotic gene is isolated then treated with reverse transcriptase to make cDNA → cDNA incorporated into plasmid (transfer vector) using restriction enzymes and DNA ligase →vector taken up by competent bacterial cells and undergo transformation → gene of interest is found using antibiotic resistance (antibiotic resistant gene attached to target gene) or color change (vectors containing genes making cells blue) methods.
36
what are the steps of PCR
denaturation at 95 degrees to separate DNA strands primer annealing at 65 degrees elongation at 70 degrees - using Taq polymerase E
37
what is used for gene therapy
virsues used to insert genes into cells bc they have the highest transduction efficiency- can cause immune response can also use naked plasmid DNA and CRISPR- less efficient
38
ESLISA
enzyme linked immunosorbent assay determine if someone has a certain antigen to diagnose disease Ab placed in microtiter plate with sample- change colour if antigen is present bc secondary antibody bound to colour with bind to the primary antibody
39
pulse chase experiment
studies gene expressoin and fate of proteins- view how protein moves through a cell pulse- aa radioactively labelled and incorporated into proteins chase- prevent radioactively labelled protein production protein tracked by simple staining
40
gel electrophoresis
separate DNA by charge and size using electric field on an agarose gel top- -ve cathode bottom- +ve anode smaller fragments travel farther to bottom
41
SDS
sodium dodecyl sulfate strong detergent used in gel electrophoersis to dentaure, linearize, and add neg charge to proteins to separate them by size and charge
42
southern blotting
Identifies fragments of known DNA sequence in a large population of DNA. Electrophoresed DNA is separated into single strands and identified via complementary DNA probes.
43
nothern blotting
Identifying fragments of known RNA using an RNA probe.
44
western blotting
Quantifies amount of target protein in a sample using sodium dodecyl sulfate polyacrylamide gel electrophoresis or SDS PAGE (proteins denatured and given negative charge proportional to their mass). Treated with primary antibody (binds to target protein) and secondary antibody (attached to indicator and binds to primary antibody).
45
FRAP
flouresence recovery after photobleaching Quantitative measure of how and where biomolecules move in a live cell. ● Protocol: Baseline fluorescence is measured → area of the sample is photobleached →photobleached molecules are replaced by unbleached molecules over time due to cell dynamics → area gradually recovers fluorescence.
46
FLIM
flouresence lifetime imaging microscopy Provides a quantitative measure of the concentration of various ions, molecules, and gases in a cell. Cells are irradiated with light and fluorescence lifetime is measured.
47
genome annotation
identifies location of a gene and coding regions in genome determines their functions
48
genomic library
stores the DNA of an organism’s genome. DNA fragments are incorporated into plasmids and can be screened for by using antibiotic resistance and color changing techniques. They can then be cloned via bacterial cloning.
49
DNA microarrays
stores the DNA of an organism’s genome. DNA fragments are incorporated into plasmids and can be screened for by using antibiotic resistance and color changing techniques. They can then be cloned via bacterial cloning.