ch 8- microscopy and lab techniques Flashcards
fixation
sticking cells to a slide
preserving them in their most lifelike state
chemical and heat fixation
what does staining do
often kills cells
electron microscopy
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
stereo microscope
a dissection microscope
view surface of object
what can you see with a compound microscope
one cell thick live cells
have poor contrast without fixing and staining
phase contrast microscopy
viewing think samples with live cells
light refracted with annular ring creating phase shift
-can lead to halo effect
how to remove halo effect
phase plate or thinner samples
what is used in flourescence microscopy
dichroic filter
- allows certain wavelengths of light to be reflected and others to pass through
distortions or artifacts decrease resolution
confocal microscopy
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
dark field micrsocopy
viewing unstained live cells bc the contrast is increased
only scattered light is viewed
scannning electron microscopy
high resolution 3D image of surface of DEHYDRATED sample
cryo SEM
sample frozen in liquid nitrogen instead of dehydrated
costly
produces artifacts
TEM
high resolution
2D images
internal structures
electron tomography
sandwiches TEM images to create 3D images of samples internal strutures
hemcytometer
counting chambers used to count cells
Gridded slide under microscope
-count cells in known area and extrapolate data
colony forming units (CFU)
estimate number of cells assuming one cell gives rise to one colony
automated cell counting
electrical resistance - counting cells by observing flow of elctricityf
flow cytometry
coutning cells by having them pass through a narrow tube and detecting them by a laser
lag phase
adaptation to prior cell division
line is horizontal, no increase in the number of bacteria
exponential (log) phase
rapid doubling
line has a positive slope
stationary phase
occurs after the exponential log phase
line is horizontal
growth rate= death rate
death phase
decline due to lack of food or other variable
negative slope
happens after stationary phase
what does differential centrifugation separate
organelles and macromolecules based on size and density
most dense to least dense organelles
nuclei
mitochondria/ chloroplasts
ER fragments
ribosomes
karyotyping
observing chromosomes under light microscope during METAPHASE
used to diagnose conditions involving chromosomal abbherations, breakages, aneuploidies
how is DNA sequencing done
dideoxy chain termination/sanger sequencing
next gen sequencing
both may use shotgun sequencing
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
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
single nucleotide polymorphisms
serve as markers for disease causing genes in humans
where do restriction enzymes cut DNA
at pallindromic sequences
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).
exonucleases
result in sticky ends by cleaving nucleotides from the ends ofpolynucleotide chain
endonucleases
cleave within a polynucleotide chain
result in either sticky or blunt ends
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
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.
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
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
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
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
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
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
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.
nothern blotting
Identifying fragments of
known RNA using an RNA probe.
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).
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.
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.
genome annotation
identifies location of a gene and coding regions in genome
determines their functions
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.
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.
