Human/Monkey TBP (TATA-box-binding protein) ELISA Kit (AEFI01101)

Key Points

The GenieColor Mycoplasma Detection kit enables the rapid detection of Mycoplasma in cell cultures by a visual pink-to-yellow color change in 1 hour without the need for specialized instrumentation.

Introduction

The GenieColor kit utilizes an advanced isothermal amplification technology to detect mycoplasma contamination in cell cultures with a pink-to-yellow visual color change. It offers several benefits including ease of use, clear results, high sensitivity, strong specificity, and anti-contamination features. By adding 1 μl of cell culture supernatant to the reaction system and incubating at 65°C for one hour, results can be visually observed. If mycoplasma contamination is present, the reaction solution changes from pink to yellow, providing a clear color contrast. Additionally, the reagent incorporates an anti-contamination system to prevent false positives from amplification product aerosols, ensuring accurate and reliable results. The GenieColor Mycoplasma Detection kit can identify 40 mycoplasma species, including 8 commonly found in cell cultures, and is compatible with a wide range of adherent cells, suspension cells, media, and serum.

Components

Storage

Store at -30°-15°C and transport at ≤0°C.

Materials Not Provided

PCR machine or water bath.

1. Collect the cell culture supernatant
   • Adherent cells: directly collect the supernatant. Cells should remain in culture for at least 72 h undisturbed prior to screening and reach 90% confluence. After 72 h, mycoplasma content in supernatant is relatively high and can be detected easily.
   • Suspension cells: collect the supernatant after centrifugation at 2,300 rpm (500 × g) for 5 min. Cells should remain in culture for at least 72 h undisturbed prior to screening. After 72 h, mycoplasma content in supernatant is relatively high and can be detected easily.
2. Preparation of reaction system
   • Thaw the GenieColor LAMP Mix from -30°-15°C and mix thoroughly.
   • Prepare the reaction system according to the following table:

• Vortex and briefly centrifuge to collect at the bottom of the tube, ensuring no bubbles in the reaction system. Add 20ul of mineral oil if using a water bath for incubation.

3. Reaction
   • Incubate at 65°C for 60 min in a PCR instrument or water bath (make sure water bath is calibrated prior to use).
4. Results
   • After the reaction is completed, observe the color in a bright environment. Pink represents negative and yellow represents positive.
   • Do not open the lid of detection tubes to prevent false positives due to aerosol contamination.

Q1: What is the sensitivity of the kit (minimum detection limit)?

• The kit can detect mycoplasma positive plasmid at a minimum concentration of 100 copies/μl.

Q2: How is accuracy ensured?

• The product includes both negative and positive controls to ensure accurate results. It has been tested against 40 different mycoplasma species, including 8 common mycoplasma contaminants.

Q3: What is the detection principle of this kit?

• The kit utilizes loop-mediated isothermal amplification (LAMP) technology. The reagent is pre-mixed with Bst isothermal amplification enzyme, mycoplasma-specific primers (inner and outer primers), and an optimized buffer. After adding the template, the LAMP reaction begins. If mycoplasma DNA is present, the reagent color changes from pink to yellow. A negative result remains pink, while a positive result turns yellow.

Q4: What sequence is used in the positive control?

• The positive control contains a plasmid with the M. hyorhinis sequence at a concentration of 10 ng/μl.

Q5: Does the positive control of the kit cause contamination?

• No, the positive control is a mycogenic plasmid, which does not cause contamination.

Q6: What does it mean if the reaction liquid color is between pink and yellow?

• A color between pink and yellow indicates a weak positive. It is recommended to repeat the test. If the repeated test is positive or weak positive, it is considered positive. If the repeated test is negative, it is considered negative.

Q7: Why does the reaction solution change color after adding the supernatant of the medium?

• The medium might interfere with the color change. If it does, then use the following treatment method:

Treatment Method:

Take a small amount of culture supernatant or cell suspension, centrifuge at 500 g for 5 minutes, and collect the supernatant. Centrifuge the supernatant at 12,000 g for 5 minutes. Aspirate 50 μl of supernatant and add 950 μl PBS. Centrifuge again, collect the mycoplasma precipitate, discard the supernatant, retain 50 μl, and use 1 μl for detection.

Q8: What is the sensitivity of the kit (minimum detection limit)?

• No, the positive control is a mycogenic plasmid, which does not cause contamination.