st PCR primer P1 2 Template (cell culture medium) 1

Usage	1. Preparation of cell culture medium: (1) Take 1 mL of cell culture medium from a cell culture in the logarithmic growth phase. (2) Centrifuge the cell culture medium at 200 g for 5 minutes at room temperature. Transfer approximately 800 μL of the culture medium to a new centrifuge tube, taking care not to carry over any cell debris. (3) Use the prepared cell culture medium as a template for nested PCR for mycoplasma detection.
Volume (μL)
2xPCR Mix	25
3xPCR Mix	4
5
6
7
8
9
2
Template (cell culture medium) 1
PCR reaction internal control 2	Add sterile ultrapure water to	3
4
5
6
7
8
9
1

PCR reaction conditions: 30 cycles, conditions are shown in the table below:

Step	Temperature	Time	Number of cycles
Pre-denaturation	center;">94℃	5min	1
Degeneration	94℃	30s	30
Annealing	55℃	30s	30
Extension	72℃	40s	30
Final extension	5min	1

（2）Take 1μL from the first PCR reaction as the template for the second PCR and perform the second PCR with primer P2. The second PCR reaction assembly (recommended on ice) is shown in the table below. The PCR reaction conditions are the same as the first PCR reaction. 43.5803%; text-align: center;">2xPCR Mix252^nd PCR Primer P22Template (1^st PCR product)1Add sterile ultrapure water to
(3) Prepare 2% agarose gel. Take 10 μL of the second PCR product for DNA electrophoresis.
3. Results The internal control of the PCR reaction is a single DNA band of 515 bp. Common mycoplasma contamination will produce a single DNA band of 236 bp-365 bp. Acholeplasma laidlawii contamination will produce double DNA bands of 426 bp and 219 bp.DescriptionThe MYCO-T™ Nested PCR Mycoplasma Detection Kit is used for qualitative detection of mycoplasma contamination in cell culture. Over 95% of mycoplasma contamination in cell culture is caused by Mycoplasma fermentans, M. orale, M. pirum, M. hyorhinis, M. hominis, M. salivariu, M. arginine, and Acholeplasma laidlawii. This kit uses nested PCR to amplify conserved gene fragments from these eight and several other common mycoplasmas in cell culture media, thereby determining the presence of mycoplasma contamination. This kit is characterized by rapid detection, high sensitivity, and high specificity. Each reaction can detect as few as 10 mycoplasma genes, with significantly improved sensitivity for Acholeplasma laidlawii, and exhibits no cross-reactivity with bacteria or cultured cells.
Product composition:
MYCO-T^TM Nested PCR Mycoplasma Detection Kit contains 2 tubes of ready-to-use primers and 1 tube of reaction internal control. The specifications are as follows:

Component	25T	50T
Primer P1	50uL	100uL
Primer P2	50uL	100uL
PCR reaction internal control	50uL	100uL

General Notes1. Hot-start Taq enzyme series is recommended for PCR reagents. The 2X PCR premix should be free of DNA gel electrophoresis stain.
2. PCR reactions should be performed in a clean bench to avoid cross-contamination and aerosol contamination. Nuclease-free pipette tips and reaction tubes should be used, and pipette tips with filters are recommended.
3. Hot-start Taq enzyme series is recommended for PCR reagents. The 2X PCR premix should be free of DNA gel electrophoresis stain.
4. PCR reactions should be performed in a clean bench to avoid cross-contamination and aerosol contamination. Nuclease-free pipette tips and reaction tubes should be used, and pipette tips with filters are recommended.Storage Temp.Store at -20℃ away from light, valid for 18 months.