bdGood genome data starts with intact, pure DNA. Aim for:
| Target | Why |
|---|---|
| High molecular weight (above 20 kb) | Needed for long reads; better libraries for short reads |
| A260/280 about 1.8, A260/230 at least 2.0 | Prevents library and sequencing failures |
| No degradation | Even genome coverage |
| Enough DNA (usually 1–2 µg) | Long-read work needs more than short-read work |
A. Blood (animals or humans)
- Collect 1–5 mL whole blood in EDTA tubes (not heparin, which inhibits PCR).
- Keep at 4 °C and extract within 24 hours, or freeze at −80 °C. Stabilising tubes (for example PAXgene) help in the field.
- Handle gently to avoid haemolysis; do not freeze before separation if you need plasma.
B. Tissue (liver, muscle, brain, leaves and others)
- Collect 25–50 mg with sterile tools; preserve as quickly as possible after dissection.
- Snap-freeze in liquid nitrogen and store at −80 °C, or place in a preservative such as RNAlater, DNA/RNA Shield or AllProtect.
- Use separate sterile tools for each sample to avoid cross-contamination.
C. Buccal swabs and saliva
- Use DNA-stabilising swabs or saliva kits (for example Oragene); swab the inner cheek for 30–60 seconds.
- No food or drink 30 minutes before. Stabilised kits keep at room temperature for weeks; dry plain swabs well or freeze at −20 °C.
D. Cell cultures and microbes
- Harvest cells by centrifugation (for example 1 million cells); for microbes, use a fresh plate or broth culture.
- Store pellets at −80 °C. Choose a lysis method suited to the cell wall (Gram type, fungi).
DNA extraction tips
- Use methods made for high-molecular-weight DNA (phenol–chloroform, MagAttract, Nanobind). For long reads, avoid column kits, which can shear DNA.
- Do not vortex or pipette up and down hard.
- Measure concentration with Qubit and purity with NanoDrop; check size on an agarose gel or TapeStation.
Common problems
| Problem | Cause | Prevention |
|---|---|---|
| Degradation | Nucleases, slow processing | Freeze at once or use a stabilising buffer |
| Low yield | Small sample, poor lysis | Enough starting material; suitable method |
| Sheared DNA | Rough handling | Gentle pipetting, no vortexing |
| RNA or protein present | Incomplete purification | RNase and proteinase K steps |
| Inhibitors | Phenol, salts, heparin | Ethanol precipitation or clean-up |
Checklist
- Sterile gloves, tubes and tips
- Collection tools (scalpel, syringe, swab) and a stabilising buffer or dry ice
- Record for each sample: ID, date, time, location and sample type
- Freeze or deliver to the lab without delay
Human samples need ethics approval and donor consent. Label tubes with codes, never with names.