Genomic DNA Isolation from Dental Plaque

DNA isolation was done according to our previously published protocol.^{21 (link)} Specifically, plaque was isolated using Chelex-100™ (Bio-Rad, USA), a styrene divinylbenzene copolymer containing paired iminodiacetate ions, which act as chelating groups in binding polyvalent metal ions.^{22 (link)} A 150 μl aliquot of suspended plaque was placed into a tube containing 10 mg Chelex 100 followed by addition of 50 μl of 120 mM Tris HCl pH 8.0 followed by addition of 10 μl of 10 mg/mL proteinase K. Proteinase K was dissolved in 30 mM Tris HCl, pH 8.0. The mix was incubated at 55 ^oC for 30 min followed by vortexing and incubation in a boiling-water bath for 8 min. Upon removal from the boiling water bath, the tubes were centrifuged at 10,000–15,000×g for 3 min and the supernatant was transferred to a clean 1.5 ml microcentrifuge tube. Prokaryotic 16S rRNA genes were amplified using universal primers (27F and 1392R) using the GemTaq kit from MGQuest (USA) (Cat# EP012). The PCR program involved a pre-amplification step of 10 cycles with annealing temperature of 56^oC followed by 20 amplification cycles with annealing temperature 58^oC. In each cycle, elongation time was 1 min 10 s, at 72^oC. PCR was finalized by extended elongation for 5 min. PCR products were purified with DNA Clean & Concentrator columns (Zymo Research, USA) and quantified using the NanoDrop (Agilent, USA).