## 4 Discussion  
The use of 100 mM NaOH to lyse bacteria and extract bacterial DNA from several different environmental samples presents an inexpensive method. Many of these extracts can be directly subjected to qPCR by fivefold dilution, where the final DNA sample is less than or equal to 1/10 th of the qPCR volume. For some samples, the sensitivity is improved through capture of the liberated DNA using silica-coated paramagnetic beads. [PERSON] et al. [46] determined that DNA has two major binding mechanisms for silica at pH > 3: through the phosphate backbone and hydrophobic binding of the DNA bases. Single-stranded nucleic acids bind to silica with greater affinity than double-stranded nucleic acids because the hydrophobic bases are exposed and because single-stranded DNA is more flexible than double-stranded DNA, maximizing contacts. Typically, nucleic acids are driven onto silica using chaotropic salts that dehydrate the nucleic acid and silica, but some amino acid buffers were also demonstrated to comparably facilitate nucleic acid binding to silica [47]. We used a recommended bead-binding buffer (BBB--see Methods) where a 50:50 mixture of 100 mM NaOH and BBB is pH 12.9. Surprisingly, some DNA will bind to the beads in 100 mM NaOH with no addition of BBB. However, more DNA is recovered from bacterial lysates through the addition of at least 0.5:1 BBB to 100 mM NaOH. Presumably, alternative binding buffers could also be compatible for DNA capture from high-pH solutions. We compared the alkaline lysis method with different rapid methods for DNA extraction from bacterial cells collected from air samples. These included rapid boiling; sonication; bead beating; enzyme digestion; and C2 minOAc. The 100 mM NaOH protocol was almost always successful for broth bacterial cultures and somewhat less successful for toothpick samples of bacterial colonies. We previously used a 10 min 100 \({}^{\circ}\)C treatment for toothpick samples of bacterial colonies with routine success, but that suffered from over- or undersampling of the colony [26,48]. The 100 mM NaOH extraction seems to be much less susceptible to \"over-load\". We also successfully used the 100 mM NaOH extraction protocol to extract DNA from samples from air, garden soil, chicken cloaca, food, environmental surfaces, sewage, pond water, and pig rectal swabs. This technique provides a high degree of reliability, has a short incubation time, and produces readily amplifiable PCR substrates. NaOH stocks are stable at ambient temperature, and the extraction requires no organic solvents, inhibitory compounds, expensive enzymes, or special equipment (shakers, mixers, heaters, etc.). Of note, assessment of an animal microbiome is related to animal nutrition and health and is a research topic of many scientists [28,39,40,49,50,51,52,53]. Efficient DNA extraction from specimens such as fecal swabs is critical to reveal microbial composition and structures. Interestingly, the paired swab microbiomes illustrated by the two DNA extraction methods, a commercial kit and the NaOH extraction, were very similar in terms of community composition and overall alpha and beta diversities. Thus, the inexpensive NaOH-magnetic bead extraction technique could be used to replace the more expensive and time-consuming commercial kit(s) for microbiome studies. One disadvantage of NaOH DNA extraction might be in converting the dsDNA to ssDNA, which requires special reagents for high-sensitivity quantification by fluorimetry and is problematic for size analysis by standard agarose gel electrophoresis. However, the liberated DNA can be easily quantified using qPCR. The NaOH method of DNA extraction has significant importance when extracting DNA from any bacterial species that cannot be cultivated, as might be present in sampling air, soil, sewage or feces. Therefore, these methods are directly applicable to sampling in resource-constrained environments.