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Mycoplasma Detection Kit PCR Method for Cell Culture

specification:50T
  • G1900-50T
  • Servicebio
  • Servicebio

Product Description

G1900-50T

Introduction:

Mycoplasma contamination can cause adverse effects on all aspects of cells, and has become a highly valued issue in cell culture. Therefore, it is very necessary to conduct regular mycoplasma contamination testing. This mycoplasma test kit adopts PCR method to specifically detect mycoplasma contamination in various cultured cell biological materials (such as cell culture, laboratory animal secretions, animal serum, etc.). The mixed primers used in the kit are specific primers designed for the conserved regions of Mycoplasma 16s rRNA sequence. Cell culture fluid can be used directly as a PCR template to specifically amplify Mycoplasma DNA with the matching Mycoplasma PCR Mix (2×), the whole process can be completed in 2 hours. This kit has high detection sensitivity and can detect as few as 20 copies of mycoplasma.

 

Storage and transportation

 Ice bag (Wet ICE) transportation; -20 ° C preservation, valid for 12 months.

 

Component Number

Component

G1900-50T

G1900-1

Mycoplasma PCR Mix(2×)

1 mL

G1900-2

Mycoplasma Primer Mix

100 μL

G1900-3

Positive Control

50 μL

G1900-4

Mycoplasma Free Water

1 mL

manual

1份

Steps

1. Sample preparation: Take an appropriate amount of the cell culture supernatant to be tested in a clean PCR tube, heat it at 95°C for 5 min using a PCR machine as a template. Serum samples can be diluted with Mycoplasma Free Water, and an appropriate amount of the sample can be taken in a clean PCR tube and heat-treated with a PCR machine at 95°C for 5 min as a template;

2. PCR system preparation: each experiment needs to set up a negative control (replace 1 μL of the sample to be tested with the same amount of Mycoplasma Free Water) and a positive control (add 0.5 μL of the positive control to the sample to be tested as a template) , Wear disposable masks and gloves during the experiment, and operate carefully to prevent improper operation from introducing exogenous mycoplasma contamination;

Component

Volume

Template

1 μL

Mycoplasma Primer Mix

2 μL

Mycoplasma PCR Mix(2×)

10 μL

Mycoplasma Free Water

To 20 μL

3. PCR program settings:

Step

Temperature

Time

Cycles

Initial Denaturation

98℃

2 min

1

Denaturation

98℃

20 s

30

Annealing

56℃

25 s

Extension

72℃

10 s

Final extension

72℃

5 min

1

Hold

4-16℃

Forever


 

4. Gel electrophoresis: Take 10 μL of PCR product and use 1% agarose gel for electrophoresis detection;

5. Result analysis: Each experiment confirms the mycoplasma contamination of the sample by comparing the test results with the negative control and the positive control. The size of the positive band is about 500 bp. If there is a band in the negative control test result, it is very likely that there is contamination in the PCR system, and it is recommended to re-experiment to confirm the result. If necessary, the PCR products can also be routinely sequenced to determine the specific Mycoplasma species.

 

Precautions

1. This kit can detect M.orale, M.arginini, M.bovis, M.fermentans, M.gallisepticum, M.hominis, M.pirum, Ureaplasma spp, M.hyorhinis, M.pneumoniae, A.laidlawii, etc. At least 11 species of mycoplasma are contaminated.

2. Thoroughly thaw and mix all reagents on ice before use, and store at -20°C after use.

3. A negative control and a positive control must be set for each experiment. The experimental group recommends setting a sample template gradient.

4. You must wear a mask during the operation and strictly follow the PCR operating standards to prevent the introduction of external pollution from affecting the experimental results.

5. In order to ensure the reliability and stability of cell experiments, it is recommended to conduct regular mycoplasma contamination testing.

6. For your safety and health, please wear lab coats and disposable gloves for operation.v

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