Ch. 5 - Resolution & Detection of Nucleic Acids Flashcards
Electrophoresis
Movement of molecules by an electric current
Neg (black) to pos (red)
Done in a matrix to contain and limit migration
DNA is a _______ charged molecule due to the phosphate groups, therefore nucleic acid moves from _______ to _______.
negatively; negative; positive
What is the min and max % of agarose that should be used for a gel?
0.5 % and 5%
_______ is used for separation of very small ssDNA.
Polyacrylamide
_______ is used for separation of dsDNA.
Agarose
What are the two function of buffers?
- To carry the current
2. To protect the sample
TBE
Tris Borate EDTA buffer
Has a larger electrical capacity (voltage)
TAE
Tris Acetate EDTA buffer
Sample runs faster
What is the function of glycerol in the loading dye?
To increase the density of the sample so it sinks to the bottom of the well and is not lost in the buffer solution
What concentration of agarose, 0.5% or 2%, is best for the separation of fragments sized anywhere from 100-500 bp?
2%
Explain the following scenarios on agarose gel:
a. No bands
b. Only molecular weight bands are visible
a. Something is wrong with the entire process, such as not staining with EtBr
b. DNA fragments weren’t loaded or the method used to produce the fragments was unsuccessful
How does PFGE separate fragments more efficiently than standard gel electrophoresis?
PFGE forces large fragments through the gel matrix by repeatedly changing directions of the electric current therefore realigning the sample
a 6% solution of acrylamide is mixed, deaerated, and poured between glass plates for gel formation. After an hour, the solution is still liquid. What might be one explanation for the gel not polymerizing?
The nucleating agent and/or the polymerization catalyst were not added to the gel solution.
A gel separation of RNA yields aberrantly migrating bands and smears. Suggest two possible explanations for this observation.
This RNA could be degraded, or improper gel conditions were used to separate the RNA.
Why does DNA not resolve well in solution without a gel matrix?
Particles move in solution based on their charge/mass ratio. As the mass of DNA increases, slowing migration, its negative charge increases, counteracting the effect of the mass.
