UA-Myco Nest PCR Mycoplasma Detection Kit
UA-Myco Nest PCR Mycoplasma Detection Kit
Product Details
Product Details
Product Specification
| Synonyms | Nested PCR Mycoplasma Detection Kit |
| Stability & Storage | Ice pack transportation. Store at -20°C away from light, valid for 18 months. |
Background
The UA-Myco nested PCR Mycoplasma Detection Kit is designed for the qualitative detection of Mycoplasma contamination in cell cultures. Over 95% of Mycoplasma contaminations in cell cultures are caused by Mycoplasma fermentans, M.orale, M.pirum, M.hyorhinis, M. hominis, M.salivariu, M.arginine, and Acholeplasma laidlawii. This kit amplifies conserved gene fragments of these eight and other common Mycoplasma species in cell culture media through nested PCR, thereby determining the presence of Mycoplasma contamination. The kit features rapid detection, high sensitivity, and high specificity. Each reaction can detect as few as 10 Mycoplasma gene copies, with particularly improved sensitivity for Acholeplasma laidlawii, and shows no cross-reactivity with bacteria or cultured cells.
Components
The Nested PCR Mycoplasma Detection Kit includes 2 tubes of ready-to-use primers and 1 tube of reaction internal control, with the following specifications:
Specification |
Components |
25T |
Primer P1 (50 μL), Primer P2 (50 μL), PCR reaction internal control (50 μL) |
50T |
Primer P1 (100 μL), Primer P2 (100 μL), PCR reaction internal control (100 μL) |
Protocol
1. Preparation of Cell Culture Medium for Testing
1) Take 1 mL of cell culture medium from a cell culture in the logarithmic growth phase.
2) Centrifuge the cell culture medium at 200g for 5 minutes at room temperature. Transfer approximately 800 μL of the supernatant to a new centrifuge tube, taking care not to transfer the pelleted cell debris.
3) Use the prepared cell culture medium as the template for nested PCR to detect mycoplasma.
2. Nested PCR Reaction
1) First PCR Reaction: Using a standard 2X PCR Master Mix as an example:
PCR Reaction Setup (Recommended to prepare on ice)
Component |
Volume ( μL） |
2xPCR Mix |
25 |
1st PCR Primer P1 |
2 |
Template (Cell Culture Medium) |
1 |
PCR Internal Control |
2 |
Add sterile ultrapure water to |
50 |
PCR Reaction Conditions: 30 cycles, see the table below for conditions
Step |
Temperature |
Time |
Cycles |
Initial Denaturation |
94℃ |
5min |
1 |
Denaturation |
94℃ |
30s |
30 |
Annealing |
55℃ |
30s |
30 |
Extension |
72℃ |
40s |
30 |
Final Extension |
72℃ |
5min |
1 |
2) Take 1 μL from the first PCR reaction as the template for the second PCR and perform the second PCR using primer P2. The setup for the second PCR reaction (recommended to prepare on ice) is shown in the table below. The PCR reaction conditions are the same as the first PCR reaction.
Component |
Volume ( μL） |
2xPCR Mix |
25 |
2nd PCR Primer P2 |
2 |
Template (1st PCR Product) |
1 |
Add sterile ultrapure water to |
50 |
3) Prepare a 2% agarose gel. Take 10 µL of the second PCR product for DNA electrophoresis.
3. Results
The PCR internal control yields a single DNA band at 515 bp. Common mycoplasma contamination produces a single DNA band between 236 bp and 365 bp, while Acholeplasma laidlawii contamination produces two DNA bands at 426 bp and 219 bp.
Guidelines
