Relic DNA FAQ

Q. Should I remove relic DNA for my study?

A: It depends. Hierarchical design principles apply here: first determine your primary research objective, then decide if relic DNA removal is necessary to achieve accurate results for that specific goal.

Whether to remove relic DNA depends on your specific research question. Our research demonstrated that removing relic DNA significantly alters microbial community composition measurements, but these changes are smaller than natural spatial and temporal variations in soil communities.

Importantly, relic DNA isn't uniformly present across environments. We found minimal amounts in highly productive soils, while less microbially active soils contained more substantial proportions.

Consider whether extracellular or dead-cell DNA would meaningfully impact your interpretation. For certain research questions (like turnover rates or active populations), removing relic DNA might be essential, while for others (like overall community surveys), it may be less critical.

Q. Do you have a more detailed Relic DNA protocol?

A: The Relic DNA protocol was developed nearly a decade ago during my postdoctoral training in the Fierer lab. While we no longer develop or use these methods in our current projects, below are some additional details.

Available Resources

  • Published methodology can be found in our papers here and here.

Equipment Notes

  • Light Source: We originally used these specialized lamps (now discontinued):

    • Activator lights from Biotium were also an option (no longer available)

    • When developing an alternative light source, you'll need to validate sufficient PMA activation. Any bright light will likely work.

  • Temperature Control: Critical for process integrity

    • All experiments maintained between 16-23°C

    • I used a cooling incubator to offset heat generated by lamps

Step-by-Step Process

  1. Preparation:

    • All steps performed in darkness until light exposure. I used a red lamp to see in the room as red wavelengths don’t activate PMA.

    • I used these transparent tubes

    • The soil to buffer ratio is crucial - 1% soil worked best for our soils since dense slurries prevent sufficient light contact. You may need to optimize this for your samples.

  2. Processing:

    • Attached tubes to moBio's vortexer adapter for 15 mL tubes

    • Placed vortexer under 650W light

    • Shaken at just over half maximum speed for ~5 minutes in darkness (allows PMA to bind to susceptible cells)

    • Exposed to 5 cycles of 30s light/30s darkness while continuing to shake. Light/dark cycling minimized heat exposure (constant heat will damage equipment)

    • Shaking during exposure ensures even light contact with suspended soil

    • After light activation, the soils are stable and can be frozen or exposed to light with no ill effect.

Additional Considerations:

  • For low biomass samples, optimization will be required

  • Testing is necessary to determine whether you have sufficient biomass

Note: These details are provided for reference purposes only. As our lab has moved on to different projects, we may not be able to provide additional support for this technique.