TABLE 2.
Overview of precipitation methods used during eDNA extraction
| Name | Chemical composition | Incubation | Extraction | Centrifugation | References |
|---|---|---|---|---|---|
| Initial ethanol | 2.2 volumes absolute EtOH + 0.1 volume 3 M NaAc | −20°C until extraction | Pelleted by centrifugation and extracted with QIAmp Tissue Extraction Kit (Qiagen) | 5500 g for 35 min at 6°C | 5 |
| −20°C until extraction | Pelleted by centrifugation and extracted with QIAmp Tissue Extraction Kit (Qiagen) | 9400 g for 1 h at 6°C | 6 | ||
| None (extracted immediately) | Pelleted by centrifugation and then either PowerWater DNA Isolation Kit (MoBio Laboratories) or QIAmp DNA Micro Kit (Qiagen) | 3220 g for 45 min at 6°C | 8 | ||
| −20°C until extraction | Pelleted by centrifugation and extracted with PCI, PowerWater DNA Isolation Kit (MoBio Laboratories), or DNeasy Blood and Tissue Kit (Qiagen) | 5500 g for 35 min at 4°C | 9 | ||
| 10–120 min on ice, then −20°C until extraction | Pelleted by centrifugation and extracted with CTAB protocol | 3220 g for 35 min at 6°C | 32 | ||
| None (extracted immediately) | Pelleted by centrifugation and extracted with Bioline Isolate II Genomic DNA Kit | 3200 g for 70 min at 6°C | 64 | ||
| −20°C until extraction | Pelleted by centrifugation and extracted with DNeasy Blood and Tissue Kit (Qiagen) | 10,000 g for 1 h at 4°C | 103 | ||
| 4°C until extraction | Pelleted by centrifugation and extracted with CTAB protocol | 3200 g for 60 min at 6°C | 11 | ||
| None (extracted immediately) | Pelleted by centrifugation, decanted, and air dried for 5 min (no EtOH pellet washes) | 3220 g for 45 min at ?°Ca | 12 | ||
| −20°C until extraction | Pelleted by centrifugation and extracted with DNeasy Blood and Tissue Kit (Qiagen) | 8500 rpm for 30 min at ?°C | 104 | ||
| −80°C until extraction | Pelleted by centrifugation and extracted with DNeasy mericon Food Kit (Qiagen) | 9000 g for 15 min at ?°C | 13 | ||
| Room temperature | Pelleted by centrifugation and extracted with DNeasy Blood and Tissue Kit (Qiagen) | 15,000 g for 15 min at 6°C | 98 | ||
| 4 and 23.9°C for 0–6 d | Pelleted by centrifugation and extracted with IBI gMAX Mini Genomic DNA Kit for blood, tissue, and cultured cells (IBI Scientific, Peosta, IA, USA). | 5000 g for 30 min at 20°C. | 13 | ||
| Terminal ethanol | 2.5 volumes 99.6% EtOH, 0.1 volume 3 M NaAc, and 50 µg tRNA3 | −20°C for 1 h | Pelleted by centrifugation, supernatant removed, one 70% EtOH wash, air dried, eluted | 20,000 g for 20 min at 4°C | 25 |
| 2 volumes 100% EtOH and 40 µl 5 M NaCl4 | −30°C overnight | Pelleted by centrifugation, supernatant removed, two 70% EtOH washes, air dried, eluted | 10,000 rpm for 30 min at 4°C | 9, 75 | |
| 2.5 volumes 100% EtOH, 0.2 volumes 5 M NaCl, and 20 µl/ml LPA5 | Room temperature (2 h) or 4°C (overnight) | Pelleted by centrifugation, supernatant removed, one or two 70% EtOH washes, air dried, eluted | 14,000 g for 30 min at 4°C | 17 | |
| 2.2 volumes 100% EtOH and 0.1 volume 5 M NaCl | −20°C overnight | Pelleted by centrifugation, supernatant removed, two 70% EtOH washes, vacuufuge and air dried, eluted | 15,000 g for 10 min at ?°C | 76 | |
| 2 volumes 100% EtOH and 0.1 volume 5 M NaCl | −20°C overnight | Pelleted by centrifugation, supernatant removed, two 70% EtOH washes, air dried, eluted | 20,000 g for 20 min at 4°C | 19 | |
| 1 ml 100% EtOH, 50 µl 3 M NaAc, and 40 µg glycogen | −20°C overnight | Pelleted by centrifugation, supernatant removed, two 70% EtOH washes, air dried, eluted | 13,000 rpm for 30 and 45 min at 4°C | 30 | |
| Initial isopropanol | 0.8 volumes 100% isopropanol and 0.1 volume 3 M NaAc | −20°C until extraction | Pelleted by centrifugation and extracted with DNeasy Blood and Tissue Kit (Qiagen) | 10,000 g for 1 h at 4°C | 103 |
| 0.8 volumes 100% isopropanol, 0.2 volumes 5 M NaCl, and ≥4.4 µg/ml glycogen | 4°C until extraction | Pelleted by centrifugation, resuspended in Lysis Buffer Ib, frozen, thawed, incubated at 50°C then precipitated again (see below) | 3270 g for 90 min at 22°Cc or 6750 g for 10 min at 22°Cd | This study | |
| Terminal isopropanol | 0.6 volumes 100% isopropanol, 0.1 volume 3 M NaAc, and 50 µg tRNA | −20°C for 1 h | Pelleted by centrifugation, supernatant removed, one 70% EtOH wash, air dried, eluted | 20,000 g for 20 min at 4°C | 25 |
| 1 volume 100% isopropanol and 0.5 volumes 5 M NaCl | −20°C overnight | Pelleted by centrifugation, supernatant removed, two 70% EtOH washes, air dried, eluted | 16,873 g for 10 min at ?°C | 16 | |
| 1.5 volumes 100% isopropanol, 0.1 volume 5 M NaCl, and 20 µl/ml LPA | −20°C for ≥2 h | Pelleted by centrifugation, supernatant removed, one or two 70% EtOH washes, air dried, eluted | 14,000 g for 30 min at 4°C | 17 | |
| 0.8 volume 100% isopropanol and 0.4 volumes 5 M NaCl | −20°C overnight | Pelleted by centrifugation, supernatant removed, two 70% EtOH washes, air dried, eluted | 15,000 g for 10 min at ?°C | 76 | |
| −4°C overnight | Pelleted by centrifugation, supernatant removed, one 70% EtOH wash, air dried, eluted | 13,000 rpm for 20 min at ?°C | 20 | ||
| 1 volume 100% isopropanol and 0.1 volume 3 M NaAc | −4°C overnight | Pelleted by centrifugation, supernatant removed, one 70% EtOH wash, air dried, eluted | 13,000 rpm for 20 min at ?°C | 89 | |
| −20°C overnight | Pelleted by centrifugation, supernatant removed, one 70% EtOH wash, eluted | Unspecified | 106 | ||
| Terminal PEG | 2 volumes 30% PEG6000 in 1.6 M NaCl, 30% PEG8000 in 0.4–2 M NaCl, and 20 µl/ml LPA | Room temperature (2 h) or 4°C (overnight) | Pelleted by centrifugation, supernatant removed, two 70% EtOH washes, eluted | 14,000 g or 20,000 g for 30, 45, or 60 min at room temp | 17 |
| 2 volumes 30% PEG8000 in 1.6 M NaCl and ≥55.5 µg/ml glycogen | 4°C overnight | Pelleted by centrifugation, supernatant removed, two 70% EtOH washes, eluted | 20,000 g for 30 min at 20°C | This study |
CTAB, cetyltrimethyl ammonium bromide; LPA, linearized polyacrylamide; NaAc, sodium acetate; NaCl, sodium chloride; PEG, polyethylene glycol; tRNA, transfer ribonucleic acid. aIndicates unspecified temperature. bSee Table 3. cSwinging-bucket rotor. dFixed-angle rotor.