Before you start
- Culture cells for at least 3 days without antibiotics before sampling.
Reagents and materials
Totals are for 6 cell lines (+10% extra). Per-cell line amounts are in the middle column.
| Reagent | Per cell line | Total |
|---|---|---|
| Culture supernatant (cells near confluence, ≥ 3 days without antibiotics) | 100 µL | 100 µL |
| PCR master mix with mycoplasma primers (kit) | 23 µL | 23 µL |
| Heat-treated supernatant (template) | 2 µL | 2 µL |
Equipment
- Heat block (95 °C)
- Thermocycler
- Agarose gel set-up
Procedure
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Collect supernatant
Take 100 µL supernatant from a dense culture.
Heat
Heat at 95 °C for 5–10 min, then spin down debris.
Set up PCR
Add 2 µL to the PCR mix; include the kit positive control, a negative (water) control and, if supplied, an internal control.
Run PCR
Run the kit’s cycling program.
Analyse
Run on an agarose gel and compare with controls; a band of the expected size indicates mycoplasma.
Critical points
- Test new lines on arrival and all lines regularly (for example monthly).
- Quarantine untested lines in a separate incubator.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Positive result | Discard the culture (preferred) or treat with an anti-mycoplasma reagent and re-test; check other lines in the same incubator. |
| Inhibited PCR | Dilute the template 1:10. |
Safety
Standard practice for your cell line’s biosafety level.
Further reading
- Uphoff CC, Drexler HG. Detection of mycoplasma contamination in cell cultures. Current Protocols in Molecular Biology (2014).
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.