TABLE 3.
Overview of buffers used during extraction of DNA from noninvasive samples
| Name | Chemical composition | Incubation | Extraction | References |
|---|---|---|---|---|
| CTAB buffer | 100 mM Tris-HCl pH 8, 1.4 M NaCl, 2% (w/v) CTAB, 20 mM EDTA, 0.25 mM PVP | 65°C for 1 h | PCIa then EtOH + precipitation | 75, 107 |
| 100 mM Tris-HCl pH 8, 1.4 M NaCl, 2% (w/v) CTAB, 0.4% (v/v) 2-ME, 1% (w/v) PVP, pH 8.0 | 50°C for 15 min | CIb then isopropanol precipitation | 108, 109 | |
| 100 mM Tris-HCl pH 8, 0.5 M NaCl, 0.5% (w/v) CTAB, 50 mM EDTA plus 2% SDS and 100 µg ProK | 55°C for 3 h | CI then EtOH precipitation with 1 mg/ml glycogen | 29 | |
| Digestion buffer | 50 mM Tris-HCl pH 8.5, 1.0 mM EDTA, 0.5% Tween-20 plus 15.9U ProK | 55°C for 15 min then 95°C for 7 min (ProK inactivation). Optional second incubation at 60°C for 10 min after addition of 150 µl digestion buffer and 7.95U ProK | DNA eluted from spin-column by centrifugation | 110 |
| Lysis buffer I | 30 mM Tris-HCl pH 8, 30 mM EDTA pH 8, 800 mM guanidium hydrochloride, 0.5% Triton X-100, pH 10 | 50°C for 1–3 h | PCI purification (optional) then EtOH, isopropanol, or PEG precipitation with 20 µg/ml LPA | 17 |
| 50°C for ≥3 h | PCI purification (optional) then PEG precipitation with ≥55.5 µg/ml glycogen | This study | ||
| Lysis buffer II | 100 mM Tris-HCl pH 8.0, 5 mM EDTA, 200 mM NaCl, 0.2% SDS, and 80 µg ProK | 55°C overnight | PCI then EtOH precipitation | 75 |
| Lysis buffer III | 10 mM Tris-HCl pH 8, 10 mM EDTA, 0.5% SDS, and 50 µg/ml ProK | 56°C for 15 min | PCI then EtOH precipitation | 33 |
| Lysis buffer IV | 50 mM Tris-HCl pH 8.0, 20 mM EDTA (pH 8.0), 150 mM NaCl, 68 mM N-lauroylsarcosine, 0.5% 2-ME, 0.33 mM DTT, and 170 µg ProK (twice) | 37°C overnight (second 170 µg ProK added immediately prior to overnight incubation) | PCI then EtOH precipitation | 111 |
| Lysis solution | 67 mM Tris-HCl (pH 8), 26.5 mM EDTA (pH 8), ∼100 mM guanidine thiocyanate, ∼228 mM trisodium phosphate dodecahydrate, and ∼34 mM NaCl, pH 9.0. For water samples, add ∼1.3% SDS (“Water Lysis Additive”) | None | Mu-DNA protocol | 41 |
| SDS buffer | 10 mM Tris-HCl, 100 mM EDTA, 200 mM NaCl, and 1% SDS | 1 mg/ml lysozyme added then 37°C for 30 min then 1 mg/ml ProK added then 55°C overnight | PCI then EtOH precipitation | 19 |
| SLB I | 50 mM Tris-HCl, 40 mM EDTA, 400 mM NaCl, 750 mM sucrose, pH 9.0 plus lysozyme (1 mg/ml), 0.5% SDS, and 160 µg ProK | 37°C for 20 min (lysozyme) then 37°C for 2 h (SDS + ProK) | PCI then EtOH precipitation | 100, 112 |
| SLB II | 50 mM Tris-HCl, 40 mM EDTA, 400 mM NaCl, 750 mM sucrose, pH 9.0 plus lysozyme (1 mg/ml), 1% SDS, and 160 µg ProK | 37°C for 20 min (lysozyme) then 37°C for 2 h (SDS + ProK) then 56°C for 15 min (phenol) | PCI then EtOH precipitation | 113 |
| SLB III | 50 mM Tris-HCl, 20 mM EDTA, 400 mM NaCl, 750 mM sucrose, pH 9.0 plus 1% SDS and 100 µg ProK | 37°C for 30 min (SDS + ProK) then 55°C for 10 min (SDS + ProK) | PCI then EtOH precipitation | 102 |
| SLB IV | 50 mM Tris-HCl, 20 mM EDTA, 400 mM NaCl, 750 mM sucrose, pH 9.0 plus lysozyme (1 mg/ml), 1% SDS, and 100 µg ProK | 37°C (lysozyme) then 55°C (SDS + ProK) | PCI purification (optional) then EtOH or isopropanol precipitation with 50 µg tRNA | 25 |
CI, chloroform:isoamyl alcohol (24:1); CTAB, cetyltrimethyl ammonium bromide; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetate; EtOH, ethanol; LPA, linearized polyacrylamide; NaCl, sodium chloride; PCI, phenol:chloroform:isoamyl alcohol (25:24:1); ProK, proteinase K; PVP, polyvinylpyrrolidone (mol wt 360,000); SDS, sodium dodecyl sulfate; SLB, sucrose lysis buffer Tris-HCl, tris hydrochloride; tRNA, transfer ribonucleic acid.