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LookOut™ Products for Mycoplasma Screening
Mynox® Mycoplasma Elimination Kit
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LookOut™ Products for Mycoplasma Screening

Mycoplasma contamination in cell culture is a serious and wide-spread problem. Detection of contamination by classical culture methods can take weeks and often cell lines are discarded. due to ineffective antibiotics typically used in clean up.. Contamination can result in lost experimental data, time, and money. Sigma-Aldrich now offer the LookOut™ series of products developed for detection, surface elimination, and culture clean-up.

LookOut™ Mycoplasma PCR Detection Kit (Product No. MP0035)

Product Description

The LookOut™ Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma and Acholeplasma contamination in cell cultures and other cell culture derived biologicals. Detection requires as little as 1 to 5 fg of mycoplasma DNA corresponding to 10 mycoplasma particles per microliter of sample.

The primer set is specific to the highly conserved rRNA operon, or more specifically, the 16S rRNA coding region in the mycoplasma genome. This allows for detection of all Mycoplasma, Acholeplasma, and Ureaplasma species tested so far and usually encountered as contaminants in cell cultures. Eukaryotic and bacterial DNA are not amplified.

Only one protocol is needed for the detection of all mycoplasma species. Because the reaction tubes included with the kit are pre-coated with appropriate dNTPs and primers, the total assay time is greatly reduced compared to general protocols that require individual loading of reaction tubes. The reaction tubes also contain DNA to serve as an internal control. For the internal control DNA, a successfully performed reaction is indicated by a distinct 481 bp band on the agarose gel.

Components

• Test Reaction Tubes, 3 strips of 8 tubes each
  (transparent tubes pre-coated with primers, dNTPs, and internal control DNA)
• Positive Control Reaction Tubes, 1 strip of 8 tubes
  (pink tubes pre-coated with primers, dNTPs, internal control DNA, and non-infectious DNA fragments of Mycoplasma orale genome, prepared by PCR)
• Mycoplasma PCR Rehydration Buffer, 1.2 ml
• Caps for PCR Tubes, 4 strips of 8 caps

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Equipment/Reagents required but not provided

• JumpStart™ Taq DNA Polymerase, Product No.D9307
• Microcentrifuge tubes, Product No. T0447
• GenElute™ Blood Genomic DNA Kit, Product No. NA2000
  (optional, for DNA extraction and purification)
• Gel Loading Solution, Product No. G7654
• 1.2% Agarose Gel

Procedure

1. Preparation of Sample Material
   Cell lines should be pre-cultured in the absence of mycoplasma active antibiotics for several days to maximize test sensitivity. Samples should be derived from cultures that are at 90-100% confluence. PCR inhibiting substances may accumulate in the medium of older cultures. For a sample from an older culture, an DNA extraction is strictly recommended prior to testing. The GenElute™ Blood Genomic DNA Kit (Product No. NA2000) is recommended.
To avoid false positive results, the use of deionized, DNA-free water, aerosol-preventive filter tips, and gloves is recommended.

Templates for PCR analysis are prepared by boiling the supernatant of cell cultures or other biologicals for 5 minutes as follows:

1. Transfer 100 µl of supernatant from the test culture to a sterile microcentrifuge tube (Product No. T0447). The lid should be tightly sealed to prevent opening during heating.
2. Boil or incubate the sample supernatant at 95 °C for 5 minutes.
3. Briefly centrifuge (5 seconds) the sample supernatant to pellet cellular debris before adding to the PCR mixture. The templates are stable at 2–8 °C for at least 1 week.


2. PCR Setup
   Total volume for each PCR is 25 µl. It is recommended to perform a positive (step 4) and a negative control reaction.
   
   
   1. Polymerase/Rehydration Buffer Preparation – Determine the total volume of Polymerase/ Rehydration Buffer required for the reactions (see Table 1). Calculations should also include an additional reaction volume (23 ml) to compensate for pipetting losses. One unit of DNA polymerase is required per reaction. For Jumpstart Taq DNA polymerase with 2.5 units/ml, a volume of 0.4 ml is required per reaction. Pipette the required volume of DNA polymerase into a clean microcentrifuge tube (Product No. T0447). Add Rehydration Buffer to bring the volume to the total required volume. Mix the Polymerase/Rehydration Buffer carefully by flicking the tube. DO NOT VORTEX!
      

      Table 1: PCR Schemes

      	Test Reaction	Positive Control	Negative Control
      Polymerase/Rehydration Buffer	23 µl	25 µl	23 µl
      Sample Volume	2 µl
      DNA-free Water			2 µl

      
      
   2. Reaction Tube rehydration - Remove strip of Test Reaction Tubes (transparent) from bag and cut off the appropriate number of tubes. Replace remaining tubes in bag and seal. Peel off protective film from tubes. Label tubes as appropriate. Add 23 ml of the prepared Polymerase/Rehydration Buffer to each test reaction tube.
   3. Sample addition - Add 2 ml of DNA-free water to the negative control and 2 ml of sample to each test reaction tube. Close tubes with Caps for PCR Tubes included in kit.
   4. Positive Control Preparation - Remove strip of Positive Control Reaction Tubes (pink) from bag and cut off the appropriate number of tubes. Replace remaining tubes in bag and seal. Peel off protective film from tubes. Label tubes as appropriate. Add 25 ml of of the prepared Polymerase/Rehydration Buffer to each tube. Close tubes with Caps for PCR Tubes included in kit.
   5. Incubation - Mix contents of each tube thoroughly by flicking tubes. DO NOT VORTEX! Incubate at room temperature for 5 minutes. Proceed immediately to thermal cycling.
   
   
3. Thermal Cycler Profile
   The programming process of your cycler is explained in the instrument manual. Two programs, a normal thermal program (std program) and a short program, are possible depending on the heating speed of your cycler.
   
   
   1. The incubation time depends on the DNA polymerase used. Hot start enzymes need to be activated at 94 °C. Please see DNA polymerase data sheet for duration.
      

      Standard Program		Short Program
      1 cycle	94 ° for 2 min 55 ° for 2 min 72 ° for 2 min	1 cycle	94 ° for 2 min
      34 cycles	94 ° for 30 sec 55 ° for 1 min 72 ° for 1 min	35 cycles	94 ° for 30 sec 55 ° for 30 sec 72 ° for 30 sec
      1 cycle	72 ° for 4 min cooldown to 4-8 °C		cool down to 4-8 °C

   
   
4. Agarose Gel
   
   1. Use 1.2 % standard agarose gel with 5 mm comb.
   2. Load 8 µl of each PCR reaction, mixed with Gel Loading Solution (Product No. G7654) per lane.
   3. Stop electrophoresis after migration of 3 cm (depending on the electrophoresis chamber used e.g., run for 20 minutes at 100 V).

Results
Gel Evaluation (Figure 1)

1. The internal control DNA and negative control samples show a distinct 481 bp band. Internal controls should appear in every lane indicating a successfully performed PCR. This band may be less intense with increased amounts of amplicons formed, caused by mycoplasma DNA loads of >5 x 10⁶ copies/ml.
2. The positive control shows a band at 270 bp and depending on the activity of the DNA polymerase used, an additional band at 481 bp due to the internal control.
3. Mycoplasma positive samples show bands in the range of 265–278 bp.

Figure 1: Relevant Amplicon Bands

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Mynox® Mycoplasma Elimination Reagent

An innovative method for fast and efficient elimination of mycoplasmas in cell cultures and virus stocks. It’s time for efficient mycoplasma elimination!

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