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UA-Myco Nest PCR Mycoplasma Detection Kit

UA-Myco Nest PCR Mycoplasma Detection Kit

Catalog Number: UA079017 Brand: UA BIOSCIENCE
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Regular price $32 USD
Regular price Sale price $32 USD
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Product Details

Product Specification


Synonyms Nested PCR Mycoplasma Detection Kit
Stability & Storage

Transported with ice packs. Store at -20℃ away from light, with a validity period of 18 months.

Background

The UA-Myco Nested PCR Mycoplasma Detection Kit is used for the qualitative detection of mycoplasma contamination in cell cultures. Over 95% of mycoplasma contamination in cell cultures is caused by Mycoplasma fermentans, M. orale, M. pirum, M. hyorhinis, M. hominis, M. salivarium, M. arginini, and Acholeplasma laidlawii. This kit amplifies the conserved gene fragments of these eight and many other common mycoplasmas in cell culture media using the nested PCR method to determine the presence of mycoplasma contamination. The kit features rapidity, high sensitivity, and high specificity. Each reaction can detect as few as 10 mycoplasma gene copies, with particularly significantly improved detection sensitivity for Acholeplasma laidlawii, and there is no cross-reactivity with bacteria or cultured cells.

Components

The nested PCR mycoplasma detection kit contains 2 tubes of ready-to-use primers and 1 tube of reaction internal control, with specifications as follows:

Specification

component

25T

One tube of Primer P1 (50 μL), one tube of Primer P2 (50 μL), one tube of PCR reaction internal reference (50 μL)

50T

1 tube of Primer P1 (100 μL), 1 tube of Primer P2 (100 μL), 1 tube of PCR reaction internal reference (100 μL)


Protocol

1. Preparation of cell culture medium to be tested
1) Take 1mL of cell culture medium from the cell culture in the logarithmic growth phase.
2) Centrifuge the cell culture medium at 200g for 5 minutes at room temperature. Transfer approximately 800μL of the medium to a new centrifuge tube, taking care not to introduce the precipitated cell debris.
3) Use the prepared cell culture medium as a template for nested PCR to detect mycoplasma.
2. Nested PCR reaction
1) First PCR reaction: Take the general 2X PCR premix reagent as an example:
PCR reaction assembly (preparation on ice is recommended))

component

Volume (μL)

2xPCR Mix                                     

25

1st PCR primer P1

2

Template (cell culture medium)

1

PCR reaction internal reference

2

Add sterile ultrapure water to                              

50

 

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

Step   

temperature

time

cyclic number

Pre-denaturation                            

94℃

5min

1

transgender               

94℃

30s

30

annealing           

55℃

30s

30

extend             

72℃

40s

30

Finally extends      

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 assembly of the second PCR reaction (preferably prepared on ice) is shown in the table below. The PCR reaction conditions are the same as those for 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 10µl of the second PCR product for DNA electrophoresis.

3. Results
The internal control of the PCR reaction shows a single DNA band at 515bp. Common mycoplasma contamination produces a single DNA band ranging from 236bp to 365bp. Acholeplasma laidlawii contamination results in double DNA bands at 426bp and 219bp.

Guidelines

It is recommended that the PCR reagents be from the hot-start Taq enzyme series. The 2X PCR premix used should not contain DNA gel electrophoresis stain.

It is recommended to set up the PCR reaction in a clean bench to avoid cross-contamination and aerosol contamination. Use nuclease-free pipette tips and reaction tubes; it is advisable to use filter tips.

Without strict verification, it is not recommended to arbitrarily change the amounts of reaction reagents.

Aliquot and store the reaction reagents according to the instructions to ensure the stability of the reagents.

This product is for research use only.