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Mycoplasma: Also known as mycoplasma, the smallest and simplest prokaryotic organism ever discovered, it is estimated that 15% to 35% of cells are contaminated with mycoplasma. Mycoplasma contamination can have many effects on cells, including metabolic, immune or biochemical properties, growth status, and cell survival, affecting the stability, reliability, and accuracy of experimental results; mycoplasma is difficult to detect, and It is difficult to eliminate, it can easily pass the 0.22μm standard filter, and it can also antagonize most antibiotics, causing huge losses to cell culture. Therefore, cells need to be tested for mycoplasma contamination on a regular basis (eg every 2 to 3 months). A number of methods are available for detecting mycoplasma, including broth or agar culture, direct or indirect fluorescent staining, ELISA, direct or indirect PCR, etc., wherein direct PCR is highly sensitive.
To ensure the accuracy of PCR results, to prevent non-specific PCR amplification and contamination, common measures are to use hot-start Taq enzyme and UDG (uracil-DNA glycosylase).
The chemically modified Taq enzyme active center is blocked such that the enzyme is not active at low temperature or normal temperature, so that non-specific amplification due to mismatching of primers is not generated prior to loading to PCR amplification. At high temperatures (such as 95 ° C), the chemically modified groups will hydrolyze off and release all enzyme activities, thus achieving the hot start of the Taq enzyme. Minimize non-specific amplification and primer dimer formation, and increase amplification sensitivity and specificity.
UDG (uracil-DNA glycosylase) can selectively hydrolyze uracil glycosidic bonds in single-stranded or double-stranded DNA containing dU to form DNA strands with deleted bases, which will further under alkaline conditions and high temperatures. The hydrolysis breaks and is thus eliminated. This product selects UDG enzyme, the optimal activity temperature is 50 °C, and it is inactivated at 95 °C.
This product contains a 2X PCR mix of chemically modified hot start enzymes, together with dUTP and UDG enzymes, to minimize non-specific amplification and primer dimer formation, and to effectively prevent cross-contamination of PCR products. At the same time, the loading of the loading Dye component is pre-added in the PCR tube, so that the pre-PCR processing and the subsequent electrophoresis loading are more convenient, so as to accurately detect the mycoplasma contamination, the reproducibility is good, and the credibility is high.