Question

You have ordered a set of enzymes that includes a protease, a DNase, and a RNase. Unfortunately, the company that you ordered them from failed to label the tubes with the specific enzymes and they are only labeled A, B and C. In your lab you have two strains of bacteria that are transformable. Strain Q is prototrophic but sensitive to the antibiotic streptomycin and strain R is auxotrophic for tryptophan but resistant to streptomycin. How might you devise an experiment to determine which tube contains DNase?

Answer 1

• 1) We have been asked to find out which of the tubes are DNAse. A, B, or C. They contain DNAse, RNAse, and Protease. We will perform a simple agarose gel assay, to determine which is the DNAse.
• In the first step, we prepare a selective LB medium containing tryptophan and streptomycin, this enables the selective cultivation of Strain R.
• We seed the medium (10 ml), let it incubate overnight at 37 degrees celsius and measure OD after 12 hours
• We then measure a drop of the culture for optical density in a spectrophotometer. A 270/280 reading of 1.7-2.0 suggests good growth
• We then employ a genomic DNA isolation kit or manual isolation such as alkaline lysis, both of which are available with standardized protocols in every laboratory. We isolate genomic DNA from our overnight culture containing strain R
• After genomic DNA isolation, we take the purified DNA and aliquot equal amounts into four vials
• We cast an agarose gel with at least 5 wells
• We then incubate three of those aliquots containing DNA with a 1:100 dilution (1 microliter) of A in one aliquot labeled A', B in B, and C, in C'. The Aliquot without any addition is a negative control.
• we load the four aliquots into four wells onto the agarose gel and the fifth will contain a DNA ladder
• An electrophoresis run is set and the gel is visualized in UV
• Let us say A contains the DNAse, lane A will have massively degraded DNA in a sheared blot like pattern, while the other lanes will have an individual discrete bar of DNA corresponding to Strain R's genomic size exactly like in the negative control lane. No digestion of DNA happens with protease and RNAse.