Wittrup: Mycoplasma Detection and Clearance

Describes the protocol for detection of mycoplasma with the PromoKine PCR Test Kit II and clearance with ciprofloxacin containing media.

Take 1.5mL of culture supernatant and transfer it to a microcentrifuge tube. Spin at 250xg for 2 minutes to pellet cellular debris. Move the new supernatant to a new sterile tube centrifuge at 15,000xg for 10 minutes to sediment mycoplasma. Aspirate all but approximately 50μL of the supernatant, enough to ensure retention of the pelleted mycoplasma. Add 50μL of the Buffer Solution and heat to 93 degrees celcius for 3 minutes. Sample can then be stored at minus 20 degrees for later use.

In a PCR tube, prepare 35uL of water, 10uL of Reaction Mix, and 5uL of the previously prepared test sample. Take appropriate precautions to prevent evaporative losses and cycle as described:

94°C 30 sec.

94°C 30 sec. | 60°C 120 sec. | 35 Cycles 72°C 60 sec. |

94°C 30 sec. 60°C 120 sec. 72°C 5 min.

On a 2% agarose gel, run 20μL of the reaction solutions without adding any sample loading buffer. Compare to a similarly prepared Positive Template Control. Stain with SYBR Gold for ~15 min. Image using Flour-S. The presence of mycoplasma species in culture is indicated by an amplified band of DNA staining at 270bp (±270 depending on the species), which will be consistent with the Positive Template Control running at 270bp.

If the cell line is found to be contaminated then the mycoplasma will need to be cleared using ciprofloxacin or another appropriate method. To clear with ciprofloxacin, supplement normal growth media with 10μg/mL cipro and continue to culture under normal conditions for ten passages. After this time, retest for mycoplasma contamination to confirm its clearance. If clearance is successful, freeze down stocks of the now clean cell line and continue to passage in normal growth media, retesting at 1, 3, and 6 months to confirm the ability to maintain mycoplasma free cultures.