Ch. 5 Resolution & Detection of Nucleic Acids
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You wish to perform an electrophoretic resolution ofyour restriction enzyme–digested DNA.
The size ofthe expected fragments ranges from 100 to 500 bp.You discover two agarose gels polymerizing on the bench.
One is 0.5% agarose; the other is 2% agarose.
Which one might you use to resolve your fragments?
The 2% agarose is best for this range offragment sizes.
After completion of the electrophoresis of DNA fragments along with the proper molecular-weight standard on an agarose gel, suppose (a) or (b) below was observed.
What might be explanations for these?
a. The gel is blank (no bands, no molecular-weightstandard).
b. Only the molecular weight standard is visible.
a. Since the molecular-weight standard is not visible, something is wrong with the general electrophoresis process. Most likely, staining with ethidium bromide was omitted.
b. The presence of the molecular-weight standardindicates that the electrophoresis and staining were performed properly. In this case, the DNA fragments were not loaded, or the method used to produce the fragments was unsucessful.
3. How does PFGE separate larger fragments more efficiently than standard electrophoresis?
PFGE forces large fragments throughthe gel matix by repeatedly changing the directionof the electric current, thus realigning the samplewith spaces in the gel matrix.
A 6% solution of 19:1 acylamide is mixed, deaerated,and poured between glass plates for gel formation.
After an hour, the solution is still liquid.
What mightbe 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.
Alternatively, improper gel conditions were used to separate the RNA.
Why does DNA not resolve well in solution (withouta 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 mass.
Why is SyBr green I less toxic than EtBr?
SyBr green is a minor groove binding dye. It does not disrupt the nucleotide sequence of DNA. EtBr is an intercalating agent that slides in between the nucleotide bases in the DNA and cancause changes in the nucleotide sequence (mutations) in DNA.
What are the general components of loading buffer used for introducing DNA samples to submarine gels?
Gel loading buffer contains a density agent to facilitate loading of sample into the wells underneath the buffer surface and a tracking dye to follow the migration of the DNA during electrophoresis.
Name two dyes that are used to monitor migration of nucleic acid during electrophoresis.
Bromphenol blue and xylene cyanol green are two of several tracking dyes.
When a DNA fragment is resolved by slab gel electrophoresis, a single sharp band is obtained.
What is the equivalent observation had this fragment been fluorescently labeled and resolved by capillary electrophoresis?
Capillary electrophoresis results will be a single peak on an electropherogram.
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