TABLE 1.
Comparative advantages and/or disadvantages of various elements of DNA extraction protocolsa
| Protocol step and option(s) | Advantage(s) and/or disadvantage(s) |
|---|---|
| Reaction vol | |
| 1.5, 2, or 10 ml | Minimized vol reduces time and reagent consumption, round bottom of 2-ml tubes complicates separation of EtOH precipitate from supernatant |
| Starting material | |
| 5 to 40 mg (wet wt) pellets | Maximum DNA yield with 20 mg, no increase with more material in 1.5-ml reaction mixture vol |
| Delipidation pretreatment | |
| Chloroform-MeOH treatment preceding lysis | Enhanced purity but substantial loss of material |
| Lysis buffer | |
| A: 5% monosodium glutamate, 50 mM Tris (pH 8.5), | |
| 25 mM EDTA | GITC lysis buffer less efficient; otherwise, no differences encountered |
| B: 15% sucrose, 50 mM (Tris pH 8.5), 50 mM EDTA | |
| C: 4 M GITC, 50 mM Tris (pH 7.2) | |
| Disruption/digestion | |
| 1 vs 10 mg/ml lysozyme, 100 vs 200 μg/ml PK | No differences encountered |
| SDS concn, 0.05 to 4% | Highest concn yields best result |
| Mechanical vs chemical lysis (BD GeneOhm kit) | Higher yields with mechanical lysis |
| High-velocity mechanical treatment | Yields efficiently increased |
| Mikro-Dismembrator S vs Precellys 24 homogenization device | Slightly enhanced yields with Precellys 24 |
| Glass, zirconia, or ceramic beads | Higher yields with glass and zirconia beads |
| 0.1-mm vs 0.5-mm beads | Higher yields with 0.1-mm beads |
| Homogenization conditions: 2-7 min at 2,000-3,000 rpm | |
| (Mikro-Dismembrator S), 2 × 40 s at 5,000 rpm to 3 × 30 s at | |
| 6,800 rpm (Precellys 24) | Harshest conditions result in highest yields |
| Purification | |
| Phenol-chloroform extraction | Purity efficiently increased |
| 2 phenol-chloroform extraction steps followed by 1 chloroform | |
| purification step only | Purity further increased |
| Phenol-chloroform vs column purification | Higher yields and purity with phenol-chloroform but less labor intensive when using columns |
| Isopropanol vs EtOH precipitation | Higher yields with isopropanol but better to handle pellet with EtOH |
| Second round of phenol-chloroform extraction and EtOH | |
| precipitation | Higher DNA quality but substantial loss of DNA material |
a
These comparisons were made with M. ulcerans strain IFIK1066089. For details of the methodology, see Materials and Methods.