Notes 3 and 4 Flashcards
Southern blotting steps
- DNA run onto gel: looks like smear
- Filter paper holds DNA
- Use probe thats highly specific to your gene of interest
- Wash away unbound probe
- Develop autoradiogram and expose to xray film
- Capture image on film
Type of filter paper used in Southern bloting
nitrocellulose
In Situ Hybridization (4 steps)
- Gently denature chromosomes that are in metaphase
- Use probe labeled with fluorescent dye
- Denature and hybridize
- Locate where it is on chromosome “the site”
Northern blotting
Transfer of RNA from a gel to a membrane
Western blotting steps
- Separate proteins on SDS gel
- Transfer to nitrocellulose membrane using electrotransfer
- This is called: Polyacrylamide gel electrophoresis
- Use antibodies to give specificity to proteins
What does SDS do?
coats proteins with a negative charge
How are proteins separated by polyacrylamide gel electrophoresis
Separated on size
No shape or charge component
Yeast model organisms
Saccharomyces cerevisiae
Schizosaccharomyces pombe
Budding yeast
Saccharomyces cerevisiae
Fission yeast
Schizosaccharomyces pombe
Slime Mold
Dictyostelium discoideum
Nematodes
Caenorhabditis elegans
Fruit fly
Drosophila melanogaster
Silk worm
Bombyx morii
Frogs
Rana pipens
Xenopus laevis
Typical dissection frog
Rana Pipens
African clawed frog
Xenopus laevis
Zebrafish
Danio rerio
Mouse
Mus musculus
Vascular plants
Arabidopsis thaliana
What’s special about wood frogs?
They’re very freeze tolerant and will freeze solid in winter
How do they prevent ice from damaging them?
Have polysaccharides to make sure ice doesn’t damage them
when the sperm and egg fuse its called
syngamy
When does the first cleavage occur?
90 minutes
When does the gastrula occur?
10 hours
20,000 cells
When does division slow (cell number)
4,096 cells
Upper side of the egg is called
Animal hemisphere
Lower side of the egg is called
Vegetal hemisphere
When it divides to four cells, those cells are called…
blastomeres
At 4,000 cells there’s the ______ stage
Blastula
What forms inside the blastula
blastocoel
What forms after the blastula?
Gastrula
When does the notochord and neurula form?
about 13 hours
When does a tail bud form?
1 day
When does a tadpole form
3 days
What is the mid blastula transition?
Where RNA synthesis occus and germ layers can develop
Mesoderm
muscle, bone
Ectoderm
skin, nervous system
Endoderm
gastro lining
How is the frog cell cycle abbreviated?
only has S & M stage
Replicate, divide
Rapidly proliferating cells
Blastomeres
5 questions of inquiry
- Observation and questions
- hypothesis
- testing the hypothesis
- Results
- Conclussion
Steps of flow cytometry
- Prepare embryonic nuclei from blastula
- Stain with propidium iodide
- excite with UV
- Quantify fluorescence by flow cytometry to determine DNA content
How is the mesoderm formed?
Contacting animal and vegetal layers
How do we break down cell layer?
CMFM (calcium magnesium free medium)
What are dispersed cells?
They aren’t touching
What are dissociated cells?
They’re touching, in a pile
What happened with dispersed cells vs. dissociated?
Dispersed cells lost their way and only stayed in S phase. Dissociated were similar to an intact embryo and still developed.
