Lab 3: DNA Spectrophotometric Quantification and Localization Using the Feulgen Stain Flashcards
What is a colorimetric method for determining the concentration of DNA in a sample?
the Dische colorimetric test.
How do you prepare a sample for the Dische test and what is the mechanism?
you must BOIL the DNA sample to hydrolyze the DEOXY sugars before doing the dische test.
mechanism: in the presence of partially hydrolyzed 2-DEOXY pentose sugars, the diphenylamine reagent turns BLUE. the BLUE color is proportional to the amount of DNA present.
What is the mechanism as to how UV spec can be used to determine the concentration of DNA in a sample?
it uses the aromaticity of the nitrogenous bases (like it used the aromaticity of Amino acids) to absorb light at the UV range.
Requirement for the UV spec to work in order to determine the conc. of DNA in a sample/
the DNA solution MUST BE DILUTE. there cannot be any turbidity.
Why do we take an A260/A280 ratio? What does this ratio tell us?
Recall in lab 1 that UV spec is good at determining the protein concentration too and thus might include DNP as part of the DNA concentrations. Nucleic acids absorb at 260, whereas proteins absorb at 280nm. Dividing the absorbance 260/280 determines the PURITY of DNA in the sample.
if ratio is over 1.85, the sample is clean of protein.
Mechanism for NanoDrop in terms of DNA quantification
Nanodrop is a type of UV spec that measures the absorbance of the aromatic nitrogenous base rings in order to measure DNA concentration. Instead of cuvettes, it uses the surface tension created by the two pedestals.
Pros and Cons of a Dische colorimetric DNA test
pros:
- relatively easy to conduct
- specific to DNA, will not detect RNA because it detects hydrolyzed DEOXY ribose sugars
cons:
-possibility of detecting other deoxypentose sugars
pros and cons of UV spec
pros;
- can be performed in dilute solutions
- no reagents need to be added
- can determine purity using a ratio
- less time consuming
cons;
- DNA CANNOT be hydrolyzed prior to conducting analysis(hyperchromatic effect)
- cannot differentiate between DNA and RNA, unlike the Dische test. RNA will still absorb at 260 nm.
What is the hyperchromatic effect?
an increase in UV absrobance values as a result of measuring DNA when it is hydrolyzed. Hydrolyzed DNA will give inaccurate readings.
T/F: Like the Dische test, you must boil the DNA sample prior to conducting UV analysis
FALSE. You need to hydrolyze DNA for the Dische test in order for the dischphenylamine reagent to bind onto the DEOXY sugars to create a blue color, but if you were to hydrolyze the DNA prior to UV analysis, it would result in erroneous hyperchromatic effect.
Pros and cons of the nanodrop experiment?
pros
- fast
- no standard curve needed
- can determine purity using ratio
- no reagents needed
cons;
- expensive
- hyperchromatic effect
- 260/280 ratio does not take RNA into account. RNA will still absorb in UV spectrum.
5 grams of wheat germ was homogenized, and 0.132 grams of DNA was extracted from it. From the total amount isolated, 25 mg of extracted DNA was placed in 25mL of 0.01 naOH to conduct a series of Dische, UV, and Nano tests. From these methods, the subsample of DNA isolated was determined to have a concentration of 0.1387 mg/ml. How much DNA per gram of wheat germ?
see notes, should be 3612.5 micrograms DNA/ gram of wheat germ.
the schiff reagent turns ___ in the presence of hydrolyzed DNA
turns red in the presence of DNA that was hydrolyzed by exposing the tissue to acid.
heterochromatin
tightly condensed DNA, will be dark red in feulgen stain
euchromatin
dispersed, less coiled DNA, will be pale red.
