Mycoplasma Detection Plus Kit (PCR)
Based on 1 Customer Validation
MCE Mycoplasma Detection Plus Kit (PCR) employs polymerase chain reaction (PCR) technology for rapid and sensitive detection of mycoplasma contamination in cultured cells and related biological samples. The kit contains a PCR premix and mycoplasma-specific primers designed against conserved regions of the 16S rRNA gene. Cell culture supernatant or cell lysates can be used directly as templates for specific amplification of mycoplasma DNA, enabling rapid identification of mycoplasma contamination.
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Storage :
-20°C, 2 years
Shipping with dry ice.
To place orders, for customer services and technical support, please contact: MedChemExpress USA
Tel: 609-228-6898 E-mail: [email protected] [email protected]
Description & Advantages
Mycoplasma are a class of prokaryotic microorganisms characterized by extremely small size, simple structure, and the absence of a cell wall, and they represent one of the most common and harmful sources of contamination in cell culture systems. Mycoplasma contamination is highly insidious; even at concentrations as high as 108 organisms/mL, no obvious changes in medium turbidity or pH are usually observed, making it difficult to detect by visual inspection or routine observation of culture conditions. Mycoplasma infection exerts significant cytotoxic effects on cultured cells and can induce a series of adverse changes, including reduced proliferation rate, abnormal morphology, chromosomal aberrations, and metabolic disorders. In severe cases, it may lead to abnormal differentiation or even cell death, thereby markedly interfering with normal cellular functions. Mycoplasma contamination not only seriously compromises the reliability, reproducibility, and consistency of experimental data, but may also directly result in experimental failure. Therefore, timely and accurate detection of mycoplasma contamination is essential to ensure cell culture quality and experimental validity.
MCE Mycoplasma Detection Plus Kit (PCR) employs polymerase chain reaction (PCR) technology for rapid and sensitive detection of mycoplasma contamination in cultured cells and related biological samples. The kit contains a PCR premix and mycoplasma-specific primers designed against conserved regions of the 16S rRNA gene. Cell culture supernatant or cell lysates can be used directly as templates for specific amplification of mycoplasma DNA, enabling rapid identification of mycoplasma contamination.
Features of MCE Mycoplasma Detection Plus Kit (PCR):
1. High sensitivity: capable of detecting as few as 20 copies of mycoplasma DNA, meeting the requirements for low-level contamination detection.
2. High specificity: the PCR system specifically amplifies mycoplasma DNA without amplifying eukaryotic or common bacterial genomic DNA, effectively minimizing non-specific interference.
3. User-friendly operation: no genomic DNA extraction is required, making it suitable for high-throughput screening of large numbers of cell samples; no heat pretreatment is required for samples such as cell culture supernatants or animal sera.
4. Rapid detection: the entire detection procedure can be completed within approximately 1 h, significantly improving efficiency.
5. Reliable results: the kit is supplied with both positive and negative controls to facilitate quality control and result interpretation, ensuring accuracy and reproducibility.
Protocol
General Protocol
1. Sample preparation
1) Adherent cells: Culture cells for 2-3 d and collect culture supernatant at approximately 80% confluence.
2) Suspension cells: Culture for 2-3 d to approximately 1 × 106 cells/mL. Centrifuge at 2,000 × g for 1 min and use the supernatant.
3) Serum may be used directly at 5 μL per reaction. Cell culture supernatant or cell lysate may be used directly for PCR without separate mycoplasma DNA extraction or heat pretreatment.
2. PCR reaction system
| Component | Test sample | Positive control | Negative control |
|---|---|---|---|
| PCR Master Mix (2×) | 15 μL | 15 μL | 15 μL |
| Primer Mix | 2 μL | 2 μL | 2 μL |
| Template | 5 μL test sample | 5 μL Positive Control | 5 μL Mycoplasma-Free Water |
| Mycoplasma-Free Water | 8 μL | 8 μL | 8 μL |
| Total volume | 30 μL | 30 μL | 30 μL |
Note: Include a positive and negative control in each run. Whenever possible, prepare PCR mixtures and add templates in separate clean areas. Wear a mask and use sterile filtered tips to reduce aerosol and environmental mycoplasma contamination.
3. PCR cycling program
| Step | Temperature | Time | Cycles |
|---|---|---|---|
| Initial denaturation | 95°C | 4 min | 1 |
| Denaturation | 95°C | 10 sec | 35 |
| Annealing | 62°C | 25 sec | 35 |
| Extension | 72°C | 10 sec | 35 |
| Final extension | 72°C | 5 min | 1 |
4. Electrophoresis and interpretation
Load 10 μL PCR product onto a 1.5% agarose gel. Compare with positive and negative controls. A specific band of approximately 254 bp indicates mycoplasma-positive material. A band in the negative control suggests contamination and the assay should be repeated.
Storage
-20°C, 2 years
Shipping with dry ice.
Attention
1. Before the experiment, ensure that all components of the kit are fully thawed and mixed well. After use, store at -20°C.
2. The serum and antibiotics in the cell culture medium do not affect the experimental results.
3. It is recommended to set up a positive control and a negative control for each experiment to ensure the accuracy of the results.
4. To avoid cross-contamination during experimental operations, it is advisable to perform them in a standard PCR area.
5. Since mycoplasmas are present in the oral cavity, wear a mask during operations to prevent sample contamination and false positive results.
6. To ensure the reliability and stability of the experiment, it is recommended to regularly check for mycoplasma contamination in cells and cell culture reagents.
7. This product is for R&D use only, not for drug, household, or other uses.
8. For your safety and health, please wear a lab coat and disposable gloves to operate.
Components
| Components | HY-K0552A-100 rxns |
|---|---|
| PCR Master Mix (2×) | 750 μL × 2 |
| Primer Mix | 200 μL |
| Positive Control | 100 μL |
| Mycoplasma-Free Water | 1 mL |
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