bdA contaminated culture gives a contaminated genome assembly. Keep the culture pure from start to finish.
1. Keep the culture pure
- Start from a single, well-isolated colony, a single spore or a hyphal-tip culture.
- Use strict aseptic technique, sterile tools, tips and fresh media; minimise exposure to the air.
- Check purity by colony appearance and microscopy; sub-culture if in doubt and discard plates with mixed growth.
- Use actively growing, healthy cultures for DNA extraction.
- Keep verified isolates as glycerol stocks or other validated stocks.
- Clean surfaces with 70% ethanol before and after work; wear gloves, mask and lab coat.
2. Extract the DNA
- Grind mycelium in liquid nitrogen or use bead-beating; CTAB-based or kit methods work well for fungi.
- Remove RNA (RNase A), proteins, polysaccharides and phenolic compounds.
- Pipette gently; do not vortex, to keep the DNA long.
3. DNA quality we need
| Parameter | Requirement |
|---|---|
| Concentration | At least 50 ng/µL |
| Total DNA | At least 1 µg |
| A260/280 | 1.8–2.0 |
| A260/230 | 2.0 or higher |
| Integrity | High molecular weight, one clear band on a gel |
| Contaminants | Free from RNA, protein, polysaccharides and phenolics |
You can also send a pure culture or mycelium and we will extract the DNA. Sequencing: Illumina NovaSeq 6000, PE150, over 300× coverage for genomes of about 20–40 Mb.