Table 1.
Characteristics of three commercial DNA isolation kits for soil and a concentration by flotation protocol following by DNA isolation as in BAM Chapter 19b, used in this study to extract genomic DNA from soil samples inoculated with 100 oocysts of Cyclospora cayetanensis.
| Flotation in Sucrose (1.12 s.d) and Fast DNATM SPIN Kit for Soil (MP Biomedicals) (Method 1) | Fast DNATM 50 mL SPIN Kit for Soil (MP Biomedicals) (Method 2) | Quick-DNATM Fecal/Soil Microbe Midiprep Kit (Zymo Research) (Method 3) | DNeasy® PowerMax® Soil Kit (Qiagen) (Method 4a—Vortexing) |
DNeasy® PowerMax® Soil Kit (Qiagen) (Method 4b—Bead-Beating) |
|
|---|---|---|---|---|---|
| Size preps in kit | 100 | 10 | 25 | 10 | 10 |
| Bead beater: yes/no; Instrument recommended | Yes FastPrep®-24 instrument with provided adapter for Fastprep (24 × 2 mL tubes) |
Yes FastPrep®-24 instrument and FastPrep® BigPrepr Adapter (2 × 50 mL tubes) |
Yes Bead beater (can be FastPrep®-24 instrument and FastPrep® BigPrepr Adapter (2 × 50 mL tubes) |
No Vortexing 10 min (vortex adaptor for 50 mL tubes (max 6 tubes). Alternatively, water bath set at 65 °C, shaking at maximum speed for 30 min |
Yes FastPrep®-24 instrument and FastPrep® BigPrepr Adapter (2 × 50 mL tubes) |
| User supplied reagents | 100% ethanol | 100% ethanol | Beta mercaptoethanol; 100% ethanol |
None | None |
| Maximum soil sample | Performed after flotation (less than 0.4 g washed material) | Up to 10 g | 5 g max (2.5 g recommended) | Up to 10 g | Up to 10 g |
| Column-based? | Yes | Yes | Yes | Yes | Yes |
| Steps in protocol | 17 | 18 | 10 | 19 | 19 (substitution of vortexing in step 4 by bead-beating) |
| Incubation times | Increase DNA yield recommended by incubation at 55 °C for 5 min | No | No | Yes, 2–8 °C for 10 min (twice) | Yes, 2–8 °C for 10 min (twice) |
| Final elution volume | 50–100 µL ** | 5 mL * | 150 µL | 5 mL * | 5 mL * |
| Cost/reaction | (625/100) $6.25 | (227/10) $22.7 | (475/25) $19.0 | (290/10) $29.0 | (290/10) $29.0 |
* After DNA isolation in Method 2 and 4a and 4b, a DNA clean and concentrator-100 protocol step (Zymo Research, catalog No. D4030) was performed. Final elution volume after clean-up was 150 µL for comparison to the other methods. After DNA isolation from all protocols, quantitative real-time qPCR was performed following BAM Chapter 19b. ** Original elution volume was 75 µL. The total elution was diluted ½ to get a final 150 µL volume for comparison to the other methods.