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Mycoplast PCR Test Kit

Kits for detecting more than 40 species of mycoplasma which include M.orale, M.arginini, M.hyorhinis ,A.laidlawii and M.Fermentans. With the trait of time-saving ,high specificity and sensitivity,it assure you a reliable experimental environment.Kits for detecting more than 40 species of mycoplasma which include M.orale, M.arginini, M.hyorhinis ,A.laidlawii and M.Fermentans. With the trait of time-saving ,high specificity and sensitivity,it assure you a reliable experimental environment.

Introduction

The PCR Mycoplasma Test Kit  which Amplification of a conserved and mycoplasma-specific 16S rRNA gene region using two specific primers ,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. The lengths and sequences of the spacer region in the16 rRNA operon which are well conserved, differ from species to species.  With PCR testing, results are obtained within three hours, since the presence of contaminant mycoplasma can be easily detected simply by verifying the bands of amplified DNA fragments of 290bp in electrophoresis. The specific primer set allows detection of many various mycoplasma species  (e.g. M. fermentans, M. hyorhinis, M. arginini, M. orale, M. salivarium, M. hominis, M. pulmonis, M. arthritidis, M. bovis, M. pneumoniae,M. pirum, M. capricolum) , with high sensitivity and specificity.

Product No.                         k0103

Kit Components (for 20 Tests)

1. Reaction Mix                     400 μl

2.Taq                                    20μl

3. Positive Template Control 20 μl

Storage: -20°C

Avoid repeated changes in the Reaction Mix temperature.

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

On dry ice for longer periods

Protocol

A. Sample preparation

Transfer 1 - 2 ml cell culture supernatant into a 2 ml centrifuge tube. To pellet cellular debris, centrifuge the sample at 250xg briefly. Transfer the supernatant into a fresh sterile tube and centrifuge at 12,000 g for 10 minutes to sediment Mycoplasma. Carefully decant the supernatant and keep the pellet . Heat to 95°C for 5 minutes.

B. PCR amplification

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

Reagents Volume

Reaction Mix          20 μl

Test sample             4 μl

Taq                          1μl

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

94°C       5min

94°C      30 secs.

60°C      30ecs,  35 cycles

72°C      45 secs.

72°C      5 min.

4 °C      Cool down

C. Analysis of amplified products by agarose gel electrophoresis

1. Apply 5 μl 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 1 % Agarose gel.

The size of DNA fragments amplified using the specific primers in this kit is 290 bp.

D. Positive Control

PCR efficiency can be checked by the use of 1 μl of Positive Template Control as a test sample. The size of the PCR product obtained using the positive template with primer pairs is 290 bp.

E. Interpretation of results

PCR products of 290bp indicate the presence of Mycoplasma DNA. Mycoplasma positive samples and Positive Template Control will result in a 290 bp product. If the Positive Template Control gives no 290 bp product this indicates failure of the PCR. Check function of the thermocycler and double-check the PCR cycler program

Note: M: DL2000 Marker

1: Negative control

2: Positive control

3-5: Different cell samples

$96.00

Freight/Packing$40.00

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