A: Yes. It works for mammalian tissue culture cells (~10⁴ cells) and single bacterial colonies (e.g., E. coli). For Gram-positive bacteria or tough cell walls, additional lysis steps (enzymatic or mechanical) may be required.
A: These steps are essential for releasing intact DNA suitable for PCR without enzymatic degradation.
- 65°C: Facilitates cell lysis and protein denaturation.
- 98°C: Ensures complete membrane disruption and nuclease inactivation.
A: Yes. For substrates that are difficult to lyse, extending the 65°C lysis step beyond 6 minutes can improve DNA recovery. Optimization is recommended for
non-standard sample types.
A: Short-term, store at -20°C. For long-term storage, store at -70°C to minimize hydrolytic damage and preserve integrity for extended periods.
A: Yes. All protocols yield PCR-ready DNA. However, for highly sensitive downstream applications such as next-generation sequencing or long-read platforms, additional cleanup (e.g., column or bead-based purification) may enhance performance.
A: Crude extracts may contain PCR inhibitors (salts, proteins, mucins, and tannins). Exceeding ~5 µl often disrupts polymerase activity, causing Ct delays, allele dropout, or complete amplification failure. Dilution or cleanup is preferable to increasing template volume.
A: Include:
- No-extract controls to monitor contamination (false positives).
- No-template controls for reagent integrity.
- Internal amplification control to detect inhibition.
For critical assays, add a short housekeeping target to assess DNA integrity and fragmentation.
