Gel Electrophoresis 🧬👹 Flashcards
What are the preferred organism for gene manipulation, and why?
Bacteria (E.Coli). It is easy to isolate DNA from bacterial cells because they have little structure (only cell wall/membrane).
What structure must be destroyed to release DNA from the cell?
Cell Membrane RAWR
lysozyme
a bacterial enzyme that digests peptidoglycan (the main component of the cell wall).
What is used to burst the cell membrane?
Detergents (ex. Sodium dodecyl sulfate) are used to disrupt the lipid bilayer and burst the cell membrane to release the intracellular components.
How are intracellular components separated once released from the cell?
Centrifugation or Chemical Extraction.
phenol
used in chemical extraction to remove unwanted proteins from DNA.
Phenol is not soluble in water, so when mixed with aqueous samples of DNA/protein, the protein will dissolve in the phenol and the nucleic acids in the water. This allows the two components to be separated with a centrifuge.
What is used to separate the RNA from the DNA after chemical extraction has taken place?
Ribonuclease (RNase)
How does RNase treatment work?
The Ribonuclease digests the RNA into ribonucleotides. This allows for the DNA to be isolated after centrifugation and the addition of alcohol.
Gel Electrophoresis
Method used to separate DNA fragments by size
Agarose
A polysaccharide extracted from seaweed that behaves like a gelatin and dissolves when heated. Solidifies into 1/4 inch thick slabs that can be used to visualize DNA.
agar agar #tedallenmoment
How does Gel Electrophoresis separate DNA molecules?
An agarose slab is submerged in a buffer-filled tank with a positive electrode on one end and a negative electrode on the other. An electrical field is applied and because of the negatively charged phosphate backbone of DNA, the DNA moves towards the positive electrode.
How does the Agarose act as a filter during electrophoresis?
The agarose slows down larger pieces of DNA, the smaller fragments will move faster and farther down the gel.
Ethidium Bromide
A dye that binds to DNA or RNA bases. The agarose gel is submerged in the tank after the current is run so that the DNA may be visualized with ultraviolet light.
carcinogenic - YuMMY
molecular weight standards
Mixture of varying sized DNA or proteins of known size used to compare to unknown proteins
Pulsed Field Gel Electrophoresis (PFGE)
Type of gel electrophoresis used for analysis of very large DNA molecules and that uses an electric field of “pulses” delivered from a hexagonal array of electrodes
