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Research Agenda Examine mouse anatomy
Choose organ/tissue for structural/histological examination
Extract DNA from mouse tissue sample
Amplify DNA in PCR to identify a gene of interest (genotype)
Research Agenda Run DNA Gel Electrophoresis to visualize genotyping results
Use bacterial transformation to express and replicate a gene of interest for in vitro studies
Check your transformation to verify gene presence using an enzyme digest assay
Examine protein localization in tissue using ImmunoHistoChemistry (IHC)
Mouse Anatomy Secure mouse on styrofoam blocks with pins Make a ventral midline incision, then cut across abdominal cavity, diaphragm and upper
chest. You will need to cut through the sternum and ribs to expose heart and lungs
Mouse Anatomy Locate the heart, lungs, diaphragm, liver, gal bladder, spleen, stomach, small intestines,
cecum, large intestine, bladder, kidneys Choose one organ to harvest for further histological examination: heart, brain, kidney, liver
Tissue Fixation By fixation we intend to:• (1) conserve the tissue from autolysis and
bacterial attack• (2) prevent the loss of cellular constituents• (3) increase optical differentiation of
cellular structures• (4) increase tissue consistency, in order to
facilitate their going through the other steps of the technique – especially slicing
How it works: Paraformaldehyde will crosslink primary amino
groups in proteins with other nearby nitrogen atoms in protein or DNA through a –CH2- linkage
A solution of 4% formaldehyde fixes pathology tissue specimens at about one mm per hour at room temperature
Depending on duration of exposure and temperature, fixation may be reversible or irreversible
Tail Snip Cut a < 5 mm tail section and
submerge it into DNA extraction buffer
Proteinase K enzyme buffer will digest tissue overnight and we will harvest the DNA tomorrow for further examination