Mycoplasma contamination will cause adverse effects on all aspects of cells, and has become a highly regarded problem in cell culture. Therefore, regular detection of mycoplasma contamination is very necessary. This Mycoplasma detection kit adopts PCR method to specifically detect mycoplasma contamination in various cultured cell biological materials (such as cell cultures, secretions of experimental animals, animal serum, etc.). The mixed primers used in the kit are specific primers designed for the conserved region of the 16S rRNA sequence of Mycoplasma. Cell culture medium can be directly used as the PCR template to specifically amplify Mycoplasma DNA. The kit can be combined with the matching Mycoplasma PCR Mix (2×) within one hour. Can detect as little as 20 copies of mycoplasma.

 

Storage and transportation

 

Wet ice transportation; Stored at -20℃, valid for 12 months.

steps
 

1. Sample preparation: Appropriate amount of cell culture supernatant to be tested was taken into a clean PCR tube, and the PCR instrument was used as a template after heat treatment at 95℃ for 5 min. Serum samples could be diluted with Mycoplasma Free Water, and appropriate samples were placed in a clean PCR tube. PCR apparatus was used for 5 min of heat treatment at 95℃ as template.

 

2. PCR system preparation: Set up negative Control (change 1 μL sample into the same amount of Mycoplasma Free Water) and Positive Control (add 0.5 μL Positive Control to 1 μL sample as Template). Wear disposable masks and gloves during the experiment. Careful operation to prevent improper operation of exogenous mycoplasma contamination;

 

4. Gel electrophoresis: take 10 μL PCR products and use 1% agarose gel for electrophoresis detection;

 

5. Result analysis: Each experiment confirmed the contamination of mycoplasma samples by comparing with the results of negative control and positive control, and the positive band size was about 500 bp. If there are bands in the negative control test results, it is likely to be contamination in the PCR system, and it is suggested to confirm the results again. If necessary, PCR products can also be routinely sequenced to identify specific mycoplasma species.

Matters needing attention

 

1. This kit can detect M. oryale, M.arginini, M. bovois, M.fermentans, M. callisepticum, M. ominis, M. Pirum, Ureaplasma SPP, M. Yorhinis, M. neumoniae, A.laidlawii, etc., at least 11 mycoplasma contamination.

2. All reagents should be thawed and mixed thoroughly on ice before use, and stored at -20℃ after use.

3. Negative control and positive control must be set for each experiment, and sample template gradient is recommended for the experimental group.

4. Masks must be worn during the operation, and PCR operation standards must be strictly followed to prevent the introduction of exogenous pollution from affecting the experimental results.

5. In order to ensure the reliability and stability of cell experiments, it is recommended that mycoplasma contamination be detected regularly.

6. For your safety and health, please wear lab coat and disposable gloves.

 

 

Products for scientific research purposes only, not for clinical diagnosis!