Lab 2: Cytochemical Localization and Extraction of DNA Flashcards
in eukaryotic cells, where is DNA found?
in the nucleus
DNA is often in the form of long, linear poly_____
polynucleotide chains
T/F: DNA is soluble in water
false. DNA is insoluble
Term for DNA and nuclear proteins combined
deoxyribonucleoprotein (DNP)
T/F: DNA and their nuclear proteins can be separated by an electric field.
false. they are very tightly complexed together and poses a problem for researchers that are wanting to study one or the other
3 main steps of DNA isolation
1) homogenization of tissue
2) centrifugation to get the insoluble material/nuclei in the form of a pellet
3) enrichment of the target molecule.
methods of enrichment to treat the nuclear pellet after centrifugation?
1) ionic strength change
2) addition of detergents
3) addition of organic solvents
4) preparation of the electric field
5) separation by filtration
T/F: DNA is soluble at physiological ph and physiological ionic strength? How did we isolate DNA in the lab?
false, DNA is insoluble at physiological pH and ionic strength. We were able to dissolve DNA in an extremely high ionic strength solution (NaCl)
outline the three steps we did in the lab to separate the DNA from DNP
1) lyse the cells and nuclei via homogenization
2) sediment the DNA into the nuclear pellet via centrifugation
3) add triton X to disturb the proteins from DNP
4) dissolve the DNA in a high salt solution. Protein will precepitate out in a high salt solution.
5) remove the precipitated protein, and then add ethanol to re-precipitate DNA
Which reagent is used in the Feulgen method of DNA localization? How is this reagent made?
uses the Schiff Reagent. Made by BLEACHING fuchsin dye.
What must you do to the tissue of analysis before you use the Feulgen method dye to visualize the DNA? What is the mechanism for visualization?
must hydrolyze the tissue in 1M HCl. Only the DNA sugard would get hydrolyzed, thus, RNA is not stained in the process.
mechanism: the bleached schiff reagent would bind to the hydrolyzed sugars and turn RED. wherever the red is seen under the microscope, there is DNA.
During the extraction of DNA, why must you blend/homogenize the dna with DNA buffer? Why can’t you use ordinary water?
- blending releases the DNA from the wheat cell’s nuclei and lyses the cells
- you need to use buffer instead of water because DNA buffer has CITRATE that chelates magnesium ions that activates DNAase activity, which prevents the breakdown of DNA. If you used normal water, DNA would be broken down once blended.
How does citrate work to preserve DNA?
citrate CHELATES and INHIBITS the magnesium ions that are supposed to activate DNAase activity whose job is to breakdown of DNA. if there were to be no citrate, DNAase would be activated due to lack of magnesium chelation, and DNA would be broken down
When you centrifuge wheat germ (after homogenization), what part of the tube would contain the DNA? (supernatant of pellet)
DNA is insoluble and thus you expect it to be n the nuclear pellet, along with nuclei, whole cells etc.
throw out the supernatant (nuclear supernatant), keep the pellet (nuclei)
how do you turn DNP —> DNA
- add NaCl to DNP. High salt concentration allows protein to dissociate from the DNA, which dissolved in the solution with high ionic strength.
- centrifuge the DNP-NaCl solution, all the protein should be seen in the pellet. the DNA would be dissolved and would be in the supernatant. COLLECT THE SUPERNATANT.
- add ethanol to the supernatant. DNA would precipitate out because DNA is insoluble in organic substances.
