BIOCHEMISTRY
Budget: $10 – $30 USD
Questions:
1. Calculate the OD260/OD280 ratio of your plasmid solution. Proteins have an absorbance
maximum at 280 nm. A DNA sample with OD260/OD280 ratio > 1.8 is considered pure.
2. What feature(s) of proteins are responsible for UV absorbance at 280 nm?
3. Another common electrophoresis buffer is TBE. How does this buffer differ from TAE?
4. Draw the structure of EDTA and how it binds metal ions. Draw the structures of the other
compound choices in this experiment, and comment on how they might prevent DNA damage.
5. What other molecules might be good antioxidants to test in the DNA nicking assay? List two
compounds, draw their structures, and hypothesize how they could prevent DNA damage.
6. What is the structure of the gel stain being used? There are several other dyes that can
bind DNA and used for gel imaging. Find an example of such a dye, and draw its structure.
7. How would the appearance of your DNA gel change if a 0.1% agarose gel slab was used?
What about a 7% agarose gel slab? For separating small fragments of DNA, is it better to use
a 7% gel or a 0.1% gel? Why?
Discuss the Following in your Writeup
I. Purity and quantity of plasmid DNA that was isolated. Discuss any potential contaminants.
Compare/contrast the methods used to quantify DNA.
II. Appearance of the gels for Cu/ascorbic acid experiments
III. Effect of chemical reagent concentration on DNA strand breaks.
IV. Effects of antioxidants tested (across the entire lab section) on DNA cleavage. Comment
on how these molecules might work to prevent DNA damage (i.e., their mechanisms of action)
1. Calculate the OD260/OD280 ratio of your plasmid solution. Proteins have an absorbance
maximum at 280 nm. A DNA sample with OD260/OD280 ratio > 1.8 is considered pure.
2. What feature(s) of proteins are responsible for UV absorbance at 280 nm?
3. Another common electrophoresis buffer is TBE. How does this buffer differ from TAE?
4. Draw the structure of EDTA and how it binds metal ions. Draw the structures of the other
compound choices in this experiment, and comment on how they might prevent DNA damage.
5. What other molecules might be good antioxidants to test in the DNA nicking assay? List two
compounds, draw their structures, and hypothesize how they could prevent DNA damage.
6. What is the structure of the gel stain being used? There are several other dyes that can
bind DNA and used for gel imaging. Find an example of such a dye, and draw its structure.
7. How would the appearance of your DNA gel change if a 0.1% agarose gel slab was used?
What about a 7% agarose gel slab? For separating small fragments of DNA, is it better to use
a 7% gel or a 0.1% gel? Why?
Discuss the Following in your Writeup
I. Purity and quantity of plasmid DNA that was isolated. Discuss any potential contaminants.
Compare/contrast the methods used to quantify DNA.
II. Appearance of the gels for Cu/ascorbic acid experiments
III. Effect of chemical reagent concentration on DNA strand breaks.
IV. Effects of antioxidants tested (across the entire lab section) on DNA cleavage. Comment
on how these molecules might work to prevent DNA damage (i.e., their mechanisms of action)