This lab was derived from: Animal Cell Culture - A Guide to Basic Techniques (5th Edition)
Operation method
Scheme 19.3 PCR Determination of Mycoplasmas
Materials and Instruments
Taq DNA polymerase Primer Positive control DNA Deoxyribonucleoside triphosphate mixture Triphosphate Agarose 1.3% TAE gel Move i. collection and preparation of dna specimens For more product details, please visit Aladdin Scientific website.
Phosphate buffer salt solution solution Tris acetate EDTA 6× spiking buffer Primer stock solution
DNA extraction and purification system DNA extraction kit for substrates Thermocycler
1. Cell lines to be tested for mycoplasma contamination should not be spiked with any antibiotics for several days prior to sample collection or at least two weeks after thawing. It should be ensured that the potency of mycoplasma in the culture supernatant is within the range of the PCR assay.
2. Collect 1 ml of culture supernatant from adherent or suspension cells. Specimens collected in this way contain live or dead cells and have the advantage that some mycoplasmas will visibly adhere to eukaryotic cells or even invade them. The supernatant can be stored at 4°C for several days or -20°C for several weeks. It should be manipulated immediately after the sample is lysed.
3. Centrifuge the supernatant at 13,000 g for 5 min and resuspend the precipitate with 1 ml of D-PBSA (D-PBSA (Phosphate Buffered Salt Solution): 137 mmol/L NaCl, 2.7 mmol/L KCl, 8.1 mmol/L Na2HPO4, 1.5 mmol/L KH2PO4, pH 7.2; autoclaved at 121°C for 20 min). ) resuspended and vortexed for mixing.
4. Centrifuge the suspension again and rinse more than once with D-PBSA as in step 3.
5. The centrifuged precipitate was resuspended with 100 μl of D-PBSA, vortexed, and then heated to 90°C for 15 min.
6. Immediately after cell disaggregation, extract DNA using standard phenol/chloroform extraction, ethanol precipitation or other DNA extraction methods.
PCR reaction
Establish rules to avoid adding too much DNA, and strictly follow the steps below:
①The place where DNA is extracted should be separated from the place where PCR is performed and the place where the gel is removed after PCR;
(ii) All reagents should be dispensed in the smallest quantities to ensure a constant supply of contaminant-free reagents;
(iii) Avoid reamplification;
④Store pipettes, tips, and tubes used only for PCR operations, and expose them to UV light frequently;
⑤ Conduct PCR operation continuously and strictly follow the steps below;
(vi) Wear gloves during the whole process of sample preparation and PCR;
(vii) The appropriate control reactions, internal control, positive control, negative control and aqueous control should be included.
1. Perform a secondary assay on each sample using the following solutions
For samples only: 1 μl NTPs, 1 μl Myco-5', 1 μl Myco-3', 1.5 μl 10× PCR buffer, 9.5 μl distilled water.
For samples and internal control DNA: 1 μl NTPs, 1 μl Myco-5', 1 μl Myco-3', 1.5 μl 10× PCR buffer, 8.5 μl distilled water, 1 μl internal control DNA, pre-mixed and stored in multiple samples, three samples were used for the reaction without internal control (including positive, negative, and aqueous reactions), and two samples were used for the reaction with internal control. The total volume should be enriched to compensate for the loss during aspiration. 
2. Add 14 μl of the mixed stock solution to a 0.2 ml PCR tube, and add 1 μl of distilled water for the aqueous control reaction.
3. Prepare the polymerase mix (10 μl for each reaction, plus an additional 10 μl to ensure adequate uptake).
Each reaction contains 1 portion of PCR buffer and 1 unit of Taq polymerase.
4. Remove all reagents used to prepare the premixed reservoir.
5. Remove the DNA sample to be tested and the positive control DNA; do not handle the reagents and samples at the same time.
6. Add 1 μl of DNA Preparation Solution to a tube that does not contain the Internal Control DNA and 1 μl of DNA Preparation Solution to a tube that does contain the Internal Control DNA.
7. To perform hot-start PCR, transfer the reaction mixture (without polymerase) to a heated reactor and perform a heating cycle as described below;
Step 1: 95°C for 7 min;
Step 2: 72°C, 3 min;
Step 3: 65°C, 2 min;
Step 4: 72°C, 5 min.
During the second step, open the heater lid and add 10 μl of polymerase mixture to each tube, the operation time may be extended if there are many samples. Opening or closing the reaction tubes should be done separately to avoid volatilization of the samples. Before proceeding to the next step in the cycle, at least 30 s should be allowed to equilibrate the temperature and remove condensate from the lid before adding Taq Polymerase to the last reaction tube and closing the heater lid.
8. After starting the first cycle, perform 32 heating cycles according to the following parameters:
Step 1: 95°C for 4 s;
The second step: 65°C, 8 s;
The third step: 72°C, 16 s;
Each cycle is additionally delayed by 1 s .
9. 72°C, 10 min Finish the final amplification step, end the reaction, and then cool the sample to room temperature.
10. Prepare a 1.3% agarose-TAE gel containing 0.3 μl of ethidium bromide/ml, cover the gel with 1× TAE (50× TAE (Tris acetate EDTA): 2 mol/L Tris base, 5.71% glacial acetic acid (V/V), 100 mmol/L EDTA, adjust the pH to 8.5), and add 12 μl of amplification product per well (10 μl of reaction mixture, 2 μl of 6× sample addition). 10 μl of reaction mixture, 2 μl of 6× sampling buffer (6× sampling buffer: 0.09% (W/V), bromophenol blue, 0.09% (W/V) xylene blue FF, 60% glycerol (V/V), 60 mmol/L EDTA)) in each well, and electrophoresis was started at 10 V/cm.
11. UV scanning was performed to show the specific products and the amplification results were recorded.