Fig. 2

DNA recovery comparison for manual and semi-automated PEG precipitation methods, and the impact of adding a non-ionic detergent (Tween-20) to the SIP gradient buffer. After a density gradient is fractionated, DNA from each fraction needs to be purified (desalted) prior to quantification and sequencing analysis. A We compared “manual” PEG precipitations of soil DNA (n=3 SIP gradients), where each fraction is precipitated in microcentrifuge tubes by an individual (as per Blazewicz et al. [17]), and semi-automated or “robot” PEG precipitations (n=3 SIP gradients), where a Hamilton STAR liquid handling robot performs the precipitations in 96-well plates. B In a separate study, we tested how adding Tween-20 to the density gradient mixture impacts DNA recovery for a large soil DNA-SIP experiment; all samples were processed semi-automatically by the robot. Tween-20 was added to a subset of the samples (+Tween, n = 38 SIP gradients) or processed using our standard density gradient buffer without Tween-20 (–Tween, n = 63 SIP gradients). Four micrograms soil DNA was used per SIP gradient, and percent DNA recovery was calculated by summing recovered DNA (measured by Picogreen) across all density fractions post-cleanup and dividing by the initial DNA input. Error bars represent the standard error of the mean