Answer to Question #342112 in Microbiology for Sam

Question

You discovered that a species of bacteria can break down StyrofoamTM (polystyrene) products due to an

enzyme it produces, polystyrenase. You wish to study the gene that codes for this enzyme.

Task 1: DNA Extraction

To begin work on the bacterium, you begin by extracting its genomic DNA (gDNA). What is the purpose of

the following procedures? Answer briefly but completely.

b. Adding RNase A and Proteinase K during extraction

Answer: __________________________________

c. Adding ethanol before recovering the DNA extract

Answer: __________________________________

Answer 1

b. First of all, you want the RNase added because it would break down contaminating RNA during your DNA isolation. And proteinase K is used during DNA extraction to digest many contaminating proteins present. It also degrades nucleases that may be present in DNA extraction and protects the nucleic acids from nuclease attack.

c. The main role of ethanol is to eliminate the solvation shell that surrounds the DNA, thus allowing the DNA to precipitate in pellet form. Additionally, ethanol helps to promote DNA aggregation. Usually, about 70 percent of ethanol solution is used during the DNA washing steps. This allows the salts to dissolve while minimizing DNA solubility. The last 100 percent ethanol wash which is mainly employed helps to promote convenient ethanol evaporation from DNA pellet, thus preventing any carryover. Ethanol is preferred to water since its dielectric constant is lower.