Mycoplasma testing (PCR-based)
Mycoplasma is a chronic, invisible cell-culture contaminant that ruins data. Test every line every 1–2 months and any time you bring in new cells from another lab.
You'll need
- Conditioned medium from a 70+% confluent culture, ≥ 24 h post-feed (no fresh medium changes)
- Mycoplasma PCR detection kit (e.g., MycoSensor PCR, Lookout, Universal Mycoplasma) OR design primers against 16S (Choppa et al. or similar)
- PCR reagents
Procedure
- Collect 1 mL conditioned medium from a culture you want to test. Cells should NOT have antibiotics in the medium for at least 1 week before testing — antibiotics suppress mycoplasma but don't eliminate it.
- Spin 5 min at 200 × g to pellet floating cells. Take the supernatant.
- Heat-treat 95 °C for 5 min to lyse mycoplasma cells. Cool. (Some kits skip this — follow the manual.)
- Use 2 µL as PCR template per kit instructions, with provided primers. Run a positive control (kit-supplied template) and an NTC.
- Run PCR program (typical: 95 °C 30 s; 35 cycles of 95 °C 15 s, 60 °C 30 s, 72 °C 30 s; 72 °C 5 min).
- Run on a 1.5% agarose gel.
Reading results
- Positive sample: 270 bp band (or kit-specific size).
- Internal control: usually a higher band confirming PCR worked.
- Positive control: must show the test band — confirms reagents are good.
- NTC: must be empty.
If you find a positive culture
- Bleach the culture and dispose; do NOT keep treating valuable lines unless you have a backup.
- Decontaminate your hood, incubator, water bath, and shared bottles.
- Treat backup vial with Plasmocin (InvivoGen) at 25 µg/mL for 2 weeks, then taper to 5 µg/mL for another 2 weeks. Re-test after 1 week off treatment.
- Inform the lab — shared reagents may be contaminated.
Prevention
- Do not share reagents (especially media bottles) between users.
- Quarantine new lines for ≥ 1 week and test BEFORE adding to the shared incubator.
- Wipe BSCs with 70% ethanol before AND after every use.
- Aliquot serum and OptiMEM — don't dip-pipette from a shared bottle.