Work Instruction wi0019 Rev: a cell Line Mycoplasma Contamination Detection with pcr materials

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Work Instruction WI0019 Rev: A

Cell Line Mycoplasma Contamination Detection with PCR

  1. Materials

    1. Mycoplasma Plus PCR Primer Set

      1. Manufacturer: Stratagene

      2. Catalog No.: 302008

    2. Taq PCR Master Mix

      1. Manufacturer: Qiagen

      2. Catalog No.: 201443

    3. Dulbecco’s Phosphate Buffered Saline (or another PBS equivalent)

      1. Manufacturer: Invitrogen

      2. Catalog: 14190-144

    4. Sterile, RNAse Free Water (Milli-Q or shipped with polymerase)

    5. PCR thermocycler

    6. UltraPure Agarose

      1. Manufacturer: Invitrogen

      2. Catalog No.: 15519

    7. TAE Buffer

      1. Composition: 20 mM Tris & 1 mM EDTA. Adjust to pH 8.0 with acetic acid.

    8. Mini Submersion Cell Electrophoresis Machine

    9. 1 kbp DNA Ladder, 0.2 µg/mL

      1. Manufacturer: Invitrogen

      2. Catalog No.: 15615-016

    10. Ethidium Bromide

    11. 6× DNA Gel Loading Buffer

      1. Composition: 30% glycerol in Milli-Q or DI water, plus 0.02% bromophenol blue.

    12. Alpha Imager for UV Transillumination & Acquisition

  1. Safety

    1. Ethidium bromide is a carcinogen. Take care to wear personal protective equipment and avoid direct contact.

  1. Procedure

    1. Getting Started

      1. Use aerosol barrier pipette tips. All sample-contact materials should be RNase, DNase-free, and sterile.

      2. Culture the cells for at least 72 hr in the absence of antibiotics prior to performing this test. This time frame is recommended by the kit manufacturer.

    2. Prepare the cell samples for PCR.

      1. Start bringing water to a boil in a beaker, thaw the Mycoplasma Plus kit.

      2. Aliquot 1mL of each cell sample into a sterile, RNAse, DNAse free microcentrifuge tube (label tubes with number 2 and up, save 1 for the positive control).

      3. Wash 2× at 300×g for 3 min in sterile PBS, resuspend in PBS.

      4. Determine the cell density: _____ cells/mL

      5. Aliquot out 50,000 cells into a new 0.5 mL microcentrifuge tube:

50,000 cells / ____ cells / mL = ____ mL

      1. Spin down and resuspend in 100 µL sterile water.

      2. Boil the tubes containing cell sample tubes for 10 minutes in water.

      3. While the samples are boiling, prepare the PCR base mixture in a test tube. Note: the Taq PCR master mix is good for 2 months at 4 ºC and should no be stored at -20 once it is thawed because repeated freeze-thaw cycles might damage the Taq DNA polymerase enzyme (there is no glycerol in the master mix).

        1. Note the total number of samples for PCR will be the number of cell samples plus two, allowing for a positive and negative control.

        2. Calculate the total volume of base mixture:

VPCR base = 50 µl/sample × ____ samples = ____ µL

        1. To a microcentrifuge tube, add enough Taq PCR master mix (2× stock):

VMaster Mix = 0.5 × VPCRbase = ____ µL

        1. Add the Mycoplasma Plus internal control

VInternal Control = 2 µL × ____ samples = ____ µL

        1. Add the Mycoplasma Plus primers

VPrimers = 2 µL × ____ samples = ____ µL

        1. Add sterile, RNAse free water

VWater = 0.5 × VPCR base - VInternal Control - VPrimers = ____ µL

      1. Briefly spin the boiled samples down in the microcentrifuge using the “short spin” setting (5 s).

      2. Vortex the StrataClean resin to mix it well (~30 s).

      3. Add 10 µL of the resin to each boiled sample and mix well by flicking.

      4. Briefly spin the resin in the sample down in the microcentrifuge using the “short spin” setting (10 s).

      5. Label the PCR tubes if needed. Save number 1 for the positive control and the last for the negative control.

      6. Add 45 µL of PCR base to each tube.

      7. Add 5 µL from the appropriate source to each tube:

        1. Tube 1: 5 µL of Mycoplasma Plus positive control

        2. Tubes 2 through N-1: Add 5 µL of sample from the appropriate cell sample source. Be sure not to add any resin.

        3. Last tube: add 5 µL of sterile RNAse free water (negative control).

    1. Run the PCR Reaction (total cycle time roughly 3 hr 40 min):

The thermocycler settings are:

Segment Number of Cycles Temp (ºC) Time (min) 1 1 94 2

  1. 2

  1. 2

2 40 94 1

50 1

72 2

    1. About 30 min before the PCR cycles complete, prepare the 2% agarose gel.

      1. Add 1.2 g of agarose to 60 mL of TAE Buffer in a glass beaker.

      2. Microwave for 1 min 30 s or until the agarose is fully dissolved.

      3. Add 10 µL of ethidium bromide.

      4. Tape the sides of the gel caster or use the casting gates in situ so the agarose can be poured in without leakage.

      5. Pour the hot agarose into the gel caster.

      6. Place the comb in the gel caster to create the gel loading points.

      7. Allow to cool, roughly 20 min.

    2. When the PCR reaction is done, load and run the gel.

      1. If needed, remove the tape and combs from the gel caster and load it and the solidified gel into the submersion gel electrophoresis immersion cell. Make sure there is enough TAE buffer in the cell to submerge the gel. Make sure the end of the gel with the combs is oriented towards the negative terminal (black) since the DNA will migrate towards the positive.

      2. Aliquot 10 µL of the PCR product for each sample into a new tube and add 2 µL of 6× DNA gel loading buffer.

      3. Add each sample to the appropriate lane.

      4. In the lane after the sample lanes, add 5 µL of 1 kbp ladder.

      5. Run the immersion electrophoresis machine with 60 V.

      6. It should take approximately 1.5 hr for the sample to complete.

    3. Image the results on the Alpha Imager.

      1. Turn on the camera power supply.

      2. Open the Fluor Chem software.

      3. Open the sample door on the Alpha Imager and lift up the light table. Place the gel directly on the UV transilluminator.

      4. Adjust the gel position if necessary to center it in the preview image.

      5. Make sure the power is activated on the transillumination bed and set the wavelength to 302 nm.

      6. Close the sample door, select filter number 2, and select UV transillumination.

      7. Adjust the camera controls to optimize the image, accessible through the top, small door. The middle of the 3 rings should adjust the focus.

      8. Make any final adjustments in the software and acquire the image.

      9. Sample negative result:


      1. Note the internal control template at 1,000 bp and the mycoplasma amplification product in the positive control sample at 874 bp.

      2. Be sure to save the resulting image in a format you can view on a different machine (e.g. tiff) and turn off the camera power supply.

  1. References

    1. Reagents

      1. Mycoplasma Plus™ PCR Primer Set Instruction Manual. Stratagene.

      2. Taq PCR Handbook. Qiagen

    2. Polymerase Chain Reaction

      1. Alberts, Bruce et al. “Manipulating Proteins, DNA, and RNA.” Molecular Biology of the Cell. New York: Taylor & Francis Group, 2002. 4th Ed. See pp. 508-509.

  1. Revision History

Date Rev Description Revision Author

4/30/08 A Added detail to materials section. Brian J. Schmidt

3/29/08 - Initial Release Brian J. Schmidt Date: 4/30/08 /4

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