| Usage | 1. Sample Preparation: Place an appropriate amount of the cell culture supernatant to be tested into a clean PCR tube and heat-treat it at 95°C for 5 minutes using a PCR instrument to use as a template. Serum samples can be diluted with Mycoplasma Free Water, and an appropriate amount of sample can be placed in a clean PCR tube. Heat-treat at 95°C for 5 minutes in a PCR instrument before use as a template.
2. PCR System Preparation: Each experiment requires a negative control (replace 1 μL of the sample to be tested with an equal amount of Mycoplasma Free Water) and a positive control (add 0.5 μL of the Positive Control to 1 μL of the sample to be tested, and both serve as the template). Wear a disposable mask and gloves during the experiment and operate with caution to prevent the introduction of exogenous mycoplasma contamination due to improper operation.
| Components | Volume | | Template | 1ul | | Mycoplasma Primer Mix | 2ul | | Mycoplasma PCR Mix (2×) | 10ul | | Mycoplasma Free Water | To 20ul |
3. PCR program settings:
| Step | Temperature | Time | Cycles | | Initial Denaturation | 98℃ | 2 min | 1 | | Denaturation | 98℃ | 20 S | 30 | | Annealing | 56℃ | 25 S | | Extension | 72℃ | 10 S | | Final extension | 72℃ | 5 min | | | Hold | 4-16℃ | Forever | |
4. Gel electrophoresis: Take 10 μL of PCR product and perform electrophoresis on 1% agarose gel; 5. Result Analysis: Each experiment is performed by comparing the sample results with those of negative and positive controls to confirm mycoplasma contamination. Positive bands are approximately 500 bp in size. If a band is present in the negative control, it is likely contamination in the PCR system. Repeat the experiment to confirm the results. If necessary, conventional sequencing of the PCR product can be performed to identify the specific mycoplasma species. |
| Description | Mycoplasma contamination can adversely affect all aspects of cells and has become a critical concern in cell culture. Therefore, regular testing for mycoplasma contamination is essential. This Mycoplasma Detection Kit uses PCR to specifically detect mycoplasma contamination in various cultured cell biological materials (such as cell culture, laboratory animal secretions, and animal serum). The primer mixture used in the kit is specifically designed to target conserved regions of the mycoplasma 16s rRNA sequence. Cell culture medium can be used directly as a PCR template to specifically amplify mycoplasma DNA. When used with the accompanying Mycoplasma PCR Mix (2 x 10), the assay is complete within one hour. This kit has high sensitivity and can detect as few as 20 copies of mycoplasma.
Product components:| Components | Name | Specifications | | A | Mycoplasma PCR Mix (2×) | 1ml | | B | Mycoplasma Primer Mix | 200ul | | C | Positive Control | 50ul | | D | Mycoplasma Free Water | 1ml |
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| General Notes | 1. This kit can detect at least 11 mycoplasma species, including M. orale, M. arginini, M. bovis, M. fermentans, M. gallisepticum, M. hominis, M. pirum, Ureaplasma spp., M. hyorhinis, M. pneumoniae, and A. laidlawii.
2. All reagents must be thoroughly thawed and mixed on ice before use. Store at -20°C after use.
3. Negative and positive controls must be performed for each experiment. A sample-template gradient is recommended for the experimental group.
4. Wear a mask during operation and strictly follow PCR protocols to prevent the introduction of exogenous contamination that may affect experimental results.
5. To ensure the reliability and stability of cell-based experiments, regular mycoplasma contamination testing is recommended.
6. For your safety and health, please wear a lab coat and disposable gloves during operation. |
| Storage Temp. | Store at -20℃, valid for 12 months. |
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