Enzymatic Digestion Followed By Heating Protocol Modifications

After looking at the Nucleic Acid Report I collected from the NanoDrop Spectrophotometer last week, I noticed that the best DNA extraction method (compared to the others) so far is the enzymatic digestion followed by heating using Ready-Lyse Lysozyme and proteinase K (Figure 1). When referring to the “best DNA extraction method”, it is the protocol that has a purity close to 1.8 for DNA and close to 2.0 for RNA under the 260/280 column (“260/280 and 260/230 Ratios”, n.d.). Because the values for the enzymatic digestion followed by heating using Ready-Lyse Lysozyme and proteinase K are somewhat close to these values (closer than the other protocols, excluding the commercial kit), during this week, I worked specifically with these two protocols. However, the past week, in tubes EDL1, EDL2 and EDL3, I added 1 microliter of Ready-Lyse Lysozyme and in tubes EDP1, EDP2 and EDP3, I added 2 microliters of proteinase K. This week, in tubes EDL 4 and EDL 5, I added 2 microliters and 3 microliters of Ready-Lyse Lysozyme respectively. Using proteinase K, in tubes EDP 4 and EDP 5, I added 3 microliters and 4 microliters of proteinase K respectively. The results of the purity (260/280 column) of DNA in each tube are shown below in Figure 2. With these results, the increase in Ready-Lyse Lysozyme seems to decrease the purity of DNA and RNA and decrease the number of nucleic acid. However, an increase in proteinase K seems to have very little effect on the amount of nucleic acid and purity of DNA and RNA, increasing slightly in both areas.

Works Cited:

260/280 and 260/230 Ratios [PDF]. (n.d.). Wilmington: Thermo Scientific.

Figure 1. Eppendorf tubes EDL1, EDL2, EDL3, EDP1, EDP2 & EDP3 used in the enzymatic digestion followed by heating protocol using Ready-Lyse Lysozyme and proteinase K.

Figure 2. Eppendorf tubes EDL4, EDL5, EDP4 & EDP5 used in the enzymatic digestion followed by heating protocol using proteinase K.