This week, I began testing DNA extraction protocols for gram positive bacteria. After reading several research papers that purported in the abstract to contain a quick and simple method for extraction, I found that these postulations were not simple enough for my purposes. Therefore, I decided to create my own protocols and test them.
Protocol #1 involved pelleting down 500µl of culture (after incubating in TSB for 24 hours @ 37°C) and then re-suspending the pellet with a 30 second vortex in a 300µl TBS/15µl 10% SDS solution. I then allowed this to sit at room temperature for 20 minutes, followed by incubation in a water bath for 10 minutes @100°C.
Protocol #2 involved pelleting down 500µl of culture (after incubating in TSB for 48 hours @ 37°C) and then re-suspending the pellet with a 30 second vortex in a 500µl sterile water solution. I then incubated the sample for 10 minutes @ 100°C.
For each protocol, after incubation @ 100°C the sample was centrifuged @12,000x g for 3 minutes. The supernatant was poured off into a 1.5 ml Eppe tube and the pellet was discarded. 4µl of supernatant and 4µl of loading dye were then loaded into 10% agarose gels with 4µl SybrGreen DNA stain added to the agarose solution before setting the gels; gels were run @100 volts for 25 minutes.
Six strains of bacteria were tested for each protocol. They were: S. aureus, B. subtilis, S. epidermidis, B. cereus, Micrococcus luteus, and E. faecalis.
Visual results under UV light indicated that each protocol produced DNA, with a larger amount present in the samples taken using protocol #1. (As I am posting this blog from offsite, however, I am unable to upload the jpegs to this blog. Gel pics to follow on a later edition.)
These results are promising for a first attempt. I will be refining and re-testing these techniques when I return to the lab next week in order to duplicate my results and potentially increase my DNA yield. Until then, have an excellent weekend.
Photo: M. luteus after gram-staining. Source: Microbeworld.org
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