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TaKaRa PCR Mycoplasma Detection Set Manual

By Dorothy Hernandez,2014-12-24 15:21
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TaKaRa PCR Mycoplasma Detection Set Manual

    EZ-PCR Mycoplasma Test Kit

    Ready-to-use PCR Mix for the detection of mycoplasma in cell culture

Cat. No.: 20-700-20 (for 20 tests)

    Lot No.:

    Expiry Date:

    Store at: -20?C

Introduction:

    EZ-PCR Mycoplasma Test Kit is designed to detect the presence of mycoplasma contaminating biological materials, such as cultured cells. Mycoplasma detection by the direct culture procedure is time-consuming and some mycoplasma species are difficult to cultivate. With PCR testing, results are obtained within a few hours, since the

    presence of contaminant mycoplasma can be easily detected simply by verifying the bands of amplified DNA fragments in electrophoresis. There is no need to prepare probes labeled with radioisotopes, or to calculate enzyme,

    dNTP’s or buffer concentrations. Instead, a ready-to-use, optimized PCR mix is supplied. The primer set allows detection of various mycoplasma species (M. fermentans, M. hyorhinis, M. arginini, M. orale, M. salivarium, M. hominis, M. pulmonis, M. arthritidis, M. bovis, M. pneumoniae, M. pirum and M. capricolum), as well as

    Acholeplasma and Spiroplasma species, with high sensitivity and specificity.

Kit Components:

    1. Reaction Mix 200µl

    2. Buffer Solution 1ml

    3. Positive Template Control 20µl

Reagents not supplied in the kit:

    1. Mineral Oil

    2. Agarose gel

    3. Distilled Sterilized water

Equipment required:

    1. Authorized thermal cycler for PCR

    2. Microcentrifuge tubes

    3. Agarose gel electrophoresis apparatus

    4. Microcentrifuge

    5. Micropipets and pipette tips (autoclaved)

Storage: -20?C

    Avoid repeated changes in the Reaction Mix temperature

    When in use, always keep the Reaction Mix on ice!

Reference:

    Rottem, S., Barile, F.M. (1993), TIBTECH, 11:143-150

Principle:

    rRNA gene sequences of prokaryotes, including mycoplasmas, are well conserved, whereas, the lengths and sequences of the spacer region in the rRNA operon (for example the region between 16S and 23S gene) differ from species to species. The detection procedure utilizing the PCR process with this primer set consists of: 1. Amplification of a conserved and mycoplasma-specific 16S rRNA gene region using two primers. 2. Detection of the amplified fragment by agarose gel electrophoresis.

    This system does not allow the amplification of DNA originating from other sources, such as cultured cells or bacteria, which affect the detection result. Amplification of the gene sequence with PCR using this primer set enhances not only the sensitivity, but also the specificity of detection. Amplified products are then detected by agarose gel electrophoresis.

Protocol:

    A. Test sample preparation:

    Transfer 0.5-1.0ml cell culture supernatant into a 2ml centrifuge tube. To pellet cellular debris, centrifuge the

    sample at 250 x g briefly. Transfer the supernatant into a fresh sterile tube and centrifuge at15,000-20,000 x g

    for 10 minutes to sediment mycoplasma. Carefully decant the supernatant and keep the pellet (the pellet will

    not always be visible). Re-suspend the pellet with 50µl of the Buffer Solution and mix thoroughly with a omicropipet. Heat to 95C for 3 minutes. The test sample can be stored at this stage at -20?C for later use.

B. PCR amplification:

    1. Prepare the reaction mixture in a PCR tube by combining the reagents shown below:

    Reagents Volume

    HO 35;l 2

    Reaction Mix 10;l

    Test sample 5;l

    2. Overlay mineral oil (approximately 40µl) to avoid the evaporation of the reaction mixture.

    3. Place all tubes in DNA thermal cycler. Set the parameters for the following conditions and perform PCR.

     94?C 30 secs.

     94?C 30 secs.

     60?C 120 secs. 35 cycles

     72?C 60 secs.

     94?C 30 secs.

     60?C 120 secs.

     72?C 5 min.

C. Analysis of amplified products by gel electrophoresis:

    1. Apply 20µl of the PCR product to the gel electrophoresis.

    2. Perform agarose gel electrophoresis with the PCR amplified samples to verify the amplified product and

    its size. Use 2% agarose gel.

    The size of DNA fragments amplified using the specific primers in this kit is 270bp.

4th. Control Template:

    By the use of 1µl of Positive Template Control as a test sample, PCR efficiency can be checked. The size of

    the PCR product obtained using the positive template with primer pairs is 270bp.

    February 2000

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