Detection of mycoplasma by PCR can be used to discriminate mycoplasma contamination of cells.
Operation method
Mycoplasma detection by PCR
Principle
The basic principle of the PCR method is the enzymatic DNA synthesis reaction, in which the DNA strand is amplified and extended by the action of DNA polymerase in the presence of a DNA template, primers and deoxyribonucleic acid.
Materials and Instruments
Cell Samples Move 1. Sample collection: Cells to be tested were cultured with double-antibody-free medium for 7d, 500μl of supernatant was taken in a sterile container and stored at 4℃ for testing. 2. Preparation of template: Under sterile conditions, take 100μl of cell culture supernatant into a sterile 0.5ml plastic centrifuge tube, cover it with a lid, and heat it at 95℃ for 5min. 3. Open the lid, add 10μl of StrataClean resin into the tube, cover the lid, vortex mixer to mix, centrifuge for 5--10s, aspirate the supernatant into a new plastic centrifuge tube, the template is made and stored at 4℃. 4. PCR reaction: The optimal conditions of the reaction system were: 10 mmol/L Tris-HCl (pH 8.38), 50 mmol/L KCl, 1.5--2.5 mmol/LMgCl2, 200 μmol/L dNTP, 2U Taq DNA polymerase; the total reaction system was 50 μl, and the reaction was carried out in 12 μl of deionized water. The total reaction system was 50 μl, and the reaction was irradiated by 12000 μJ/cm2 UV lamp with deionized water. (1) Add 35.2 μl of deionized water and 5 μl of 10×Taq reaction buffer to a 0.5 ml plastic centrifuge tube. (2) Add the following components sequentially: 0.4 μl dNTP (25 mmol/L); 0.4 μl Taq enzyme (5 U/μl); 2 μl primer. (3) Add 2 μl deionized water for a total volume of 45 μl. (3) Add 2μl deionized water, total volume 45μl. (4) Add 5μl of made template to the reaction system. (5) Add 5 μl each of positive control and internal control to the respective reaction system. (6) Take 1 centrifuge tube containing the above reaction system and add 5μl of deionized water as a negative control tube. (7) Add 100 μl of mineral oil to the reaction system. (8) PCR program Program 1: 94℃ 2min, 50℃ 2min, 72℃ 2min, 1 cycle; Program 2: 94℃ 1min, 50℃ 1min, 72℃ 2min, a total of 40 cycles. 5. Agarose gel electrophoresis: after the PCR reaction, agarose gel electrophoresis was carried out with 2% agarose gel concentration. After the end of electrophoresis, the results were analyzed by gel imaging. 6. Experimental results and analysis: This method is a qualitative method for detecting mycoplasma, in the electrophoresis lane, Marker (DNA molecular quality standard reference), positive control, and internal control will appear different electrophoretic bands. A sample is considered to be contaminated with mycoplasma when a bright band appears in the lane of the sample being tested and is positioned between the positive and negative control bands. Sometimes multiple bands are found in one lane, which may be due to the sample being infected with more than two mycoplasma species. Caveat 1. Pre-operation of PCR reaction should be carried out in a sterile environment. 2. Before detection, the cells to be tested should be cultured with double-antibody-free medium for 7d. Common Problems Source Experimental Techniques in Cell Biology For more product details, please visit Aladdin Scientific website.
dNTP TapDNA Polymerase Agarose Mineral Oil Mycoplasma Detection Kit
Ultra-clean bench PCR instrument Electrophoresis instrument Gel imaging analysis system Benchtop centrifuge Vortex mixer