bdWhole genome sequencing needs pure, intact, high-molecular-weight DNA free from RNA, protein and inhibitors. This matters for short reads (Illumina) and even more for long reads (Nanopore, PacBio).
1. Grow the culture
- Inoculate 5–10 mL broth (LB, TSB or another suitable medium) with one fresh, single colony.
- Incubate at the right temperature (for example 37 °C) with shaking at 200 rpm for 12–18 hours, to mid- or late-log phase (OD600 about 0.6–1.0). Avoid overgrowth.
2. Harvest the cells
- Centrifuge at 6,000–10,000 × g for 5–10 minutes and discard the supernatant.
- Recommended: resuspend in 1× PBS or TE, spin again and discard, to remove medium that may contain inhibitors.
3. Extract the DNA
| Platform | DNA needed | Suggested method |
|---|---|---|
| Illumina (short reads) | Clean, moderate size | Silica column kits (for example Qiagen, Promega) |
| Nanopore / PacBio (long reads) | Very pure, above 30 kb | Phenol–chloroform or HMW kits (MagAttract HMW, Nanobind) |
- Gram-positive bacteria: lysozyme (20 mg/mL) at 37 °C for 30–60 minutes.
- Proteinase K + SDS to lyse cells and digest proteins; add RNase A.
- Purify by phenol–chloroform or magnetic beads.
- Ethanol-precipitate to concentrate if needed. Do not vortex; pipette gently.
4. Check quality before sending
| Check | Tool | Target |
|---|---|---|
| Concentration | Qubit (dsDNA assay) | At least 50 ng/µL and 1 µg in total |
| Purity | NanoDrop | A260/280 1.8–2.0; A260/230 at least 2.0 |
| Integrity | 0.8% agarose gel or TapeStation | One high-molecular-weight band, little smearing |
Troubleshooting
| Problem | Cause | Solution |
|---|---|---|
| Degraded DNA | Harsh pipetting, old culture, nucleases | No vortexing, fresh culture, work on ice |
| Low yield | Poor lysis, little biomass | More culture; lysozyme for Gram-positives |
| RNA contamination | No RNase step | Add RNase A |
| Protein or phenol carry-over | Incomplete clean-up | Extra ethanol wash or column clean-up |
| Inhibitors | Medium carry-over, polysaccharides | Wash the pellet; bead clean-up |
You can also send a pure culture or cell pellet and we will extract the DNA. A contaminated culture gives a contaminated genome, so always start from a single, verified colony.