Removal of mycoplasma

As a good practise DO NOT use antibiotics (PenStrep) in the culture medium, but rather work with sterile conditions. In the presense of antibiotics, the few resistent bacteria are still a powerful source of mycoplasma.
To remove mycoplasma from your culture use the reagent from ICN (mycoplasm removal agent, #30-500-44). Treat the cells for three passages with the agent.

Mycoplasma detection by PCR

The presence of mycoplasma is tested by PCR with the culture supernatant and primers specific for a mycoplasma gene (rRNA). The size of the amplicon ist 271 bp.

Materials required:

  1. Taq polymerase with 10x buffer (make sure that the preparation of the Taq itself does not contain mycoplasma. If this is the case use commerical Taq.
  2. PCR water
  3. dNTP mix (each 2,5 mM)
  4. 5' primer: GGA GCA AAC AGG ATT AGA TAC CCT
  5. 3' primer: TGC ACC ATC TGT CAC TCT GTT AAC CTC
  6. positive control (e.g. PCR product from a previous PCR)
  7. supernatant from a confluent culture, not treated with antibiotics for at least two weeks

reaction mix (50ul):

    36,5 ul H2O
    5 ul 10xPCR buffer with Mg2+
    4 ul dNTP mix (2,5 mM each)
    2 ul primer mix (each 10 uM)
    0.5 ul Taq polymerase (1 U)
    2 ul medium from cell culture

PCR programme:

    3 min 94¡C
    ---
    30 sec 94¡C
    1 min 55 ¡C
    1 min 72¡C
    repeat 35x
    ---
    4 min 72¡C

Procedure:

  1. use filter tips for pipetting
  2. prepare a master mix (without Taq and medium)
  3. pipette medium into labelled PCR tubes
  4. add polymerase to master mix, mix
  5. distribute master mix to PCR tubes (48 ul each)
  6. put PCR tubes to PCR maschine and start the programme
  7. analyse by agarose gel electrophoresis (2%), use 10 ul