Universal Mycoplasma Detection Kit

Universal Mycoplasma Detection Kit

Catalog Number 30-1012K (40 Assays)
Instruction Sheet
Store at -20°C.
Gel Electrophoresis Protocol
This product is intended for laboratory research purposes only. It is not
Step intended for clinical use.
1 Prepare a 3% agarose gel.
2 Prepare samples: Add 10 µL of the PCR product to 1.5 µL loading buffer. Mix thoroughly. Introduction
3 Load samples and a DNA marker (e.g., 100 bp ladder) onto the gel. The Universal Mycoplasma Detection Kit offers a quick and sensitive PCR-based test to detect myco-
plasma contaminants in cell culture. All components required for the PCR reaction are provided and have
4 Electrophorese until the tracking dye migrates 60-70% the length of the gel.
been optimized for amplification. High specificity is obtained through the utilization of a proprietary mix
5 Stain the gel with ethidium bromide or similar stain and view with UV illumination. of buffers, dNTPs and thermostable polymerase, combined with universal primers that are specific to the
Results: A test sample that is positive for the presence of mycoplasma shows a distinct band at 434 16S rRNA coding region in the mycoplasma genome. DNA originating from other sources, such as tissue
to 468 bp. The positive control samples exhibit a 464-bp band. There should be no visible band in the samples or [Link], is not amplified. A touchdown PCR regimen increases sensitivity of the assay, along with
negative control lane. enhancing specificity.

Detection of Top 8 Mycoplasma Species

The kit detects over 60 species of Mycoplasma, Acholeplasma, Spiroplasma and Ureaplasma, including the
Detection of the top eight mycoplasma
top eight species most likely to afflict cell cultures: M. arginini, M. fermentans, M. hominis, M. hyorhinis, M.
species that infect cell cultures is shown.
Distinct bands in the 434 bp to 468 bp orale, M. pirum, M. salivarium, and A. laidlawii. Samples that are positive for mycoplasma are easily recog-
range confirm the presence of myco- nized by a distinct PCR product ranging in size from 434 to 468 bp on an agarose gel.
plasma.
Kit Components
The first lane is a 100 bp DNA ladder with
a highlighted band at 500 bp. The second Component Volume Composition Storage
lane is 2.5 pg of the positive control (M. Lysis Buffer 2 mL Lytic agent + digestive enzymes -20°C
arginini chromosomal DNA) displaying a
464-bp PCR product. Cultures contami- Universal PCR Mix 0.8 mL Proprietary mix of buffers, dNTPs, thermostable poly- -20°C
nated with mycoplasma typically generate merase
signals similar to the positive control and
Universal Primers 0.1 mL Proprietary mix of universal forward and reverse primers -20°C
at least as strong as those shown here.
Sample Lysis Tubes 40 each 2-mL snap cap tubes, tight seal -20°C
Troubleshooting* Positive Control 50 µL 1 pg/µL pUC19:: [Link] target in TE -20°C
Problem Potential Cause Solution
Positive control does not PCR did not work. Check to make sure the touchdown protocol was Quality Control Specifications
exhibit a 464-bp band. programmed correctly in the thermal cycler.
Limit of detection (LOD): < 20 genomes of [Link] and A. laidlawii are detected in a standard assay. The
Negative control lane shows Contamination during Prepare new samples and repeat PCR. If possible, use range of detection varies depending on species, cell type, media and state of cell growth. See [Link].
a 464-bp band. preparation of the PCR a dedicated PCR work station with laminar flow or a
samples. laminar flow hood to avoid environmental contamina- org for detection results on over 60 mycoplasma species.
tion.
A Certificate of Analysis is available upon request for each lot of the Universal Mycoplasma Detection Kit. The
No bands present in the Inhibition of PCR by the cell If more than 105 cells were used, thaw cell extract and MSDS is available upon request.
sample or positive control lysate, which suggests that dilute to 105 cells per 50 µL with Lysis Buffer. If the
+ lysate lanes, but a 464-bp too many cells were used in number of cells is unknown, then dilute the extract
band observed in the positive the assay. 1 to 5 and 1 to 10 with Lysis Buffer. Repeat the lysis Equipment and Materials Required but not Included in the Kit
control lane. step in the Sample Preparation Protocol. Perform PCR
with 2.5 µL of the diluted extracts. Microcentrifuge Thermal cycler and PCR tubes
Bands outside the 434 to 468 Non-specific bands that These bands do not indicate mycoplasma contamina- Heating blocks for microcentrifuge tubes at 37°C Agarose gel electrophoresis apparatus and
bp range are observed in the occasionally form during tion. No action required. and 95°C buffers
PCR products. extended PCR cycles.
Positive-displacement pipette and aerosol-resistant Gel loading dye and DNA stain (ethidium
* For further information on mycoplasma and mycoplasma detection, please visit [Link] or tips bromide)
contact tech@[Link] or your local distributor.

American Type Culture Collection IV-6326 05.05.16 800-638-6597 (U.S., Canada, and Puerto Rico) American Type Culture Collection 800-638-6597 (U.S., Canada, and Puerto Rico)
P.O. Box 1549 703-365-2700 P.O. Box 1549 703-365-2700
Manassas, VA 20108 USA Page 4 of 4 Fax: 703-365-2750 Manassas, VA 20108 USA Fax: 703-365-2750
[Link] E-mail: tech@[Link] [Link] Page 1 of 4 E-mail: tech@[Link]
© 2015 ATCC. All rights reserved. Or contact your local distributor Or contact your local distributor
 Universal Mycoplasma Detection Kit Universal Mycoplasma Detection Kit
Instruction Sheet Instruction Sheet

Sample Preparation Protocol

Samples should be derived from cell cultures that are 50% to 70% confluent. Use of more than 106 cells
per sample may inhibit PCR or result in samples that are not homogeneous.
Step 2 Prepare the reaction mixtures in PCR tubes as follows:
1 Cell Harvest: Component Test Samples Positive Positive Control + Negative
A. Suspension cells: Count cells. 104 - 105 cells are needed for the assay. Control Test Sample* Control
B. Adherent cells: Scrape the cells into the existing culture media and suspend. Do not treat
Universal PCR Mix + 22.5 µL 22.5 µL 22.5 µL 22.5 µL
cells with trypsin or EDTA as these agents disrupt mycoplasma.
Primers Mix
2 Transfer 1 mL cell suspension (104 to 105 cells) into the Sample Lysis Tubes and centrifuge at
Test Sample 2.5 µL ---- 2.5 µL ----
13,000 rpm for 3 minutes at 4°C. (Note: These tubes were selected for use because they resist
Positive Control ---- 2.5 µL 1.0 µL ----
opening during the inactivation step 6).
H2O or TE ---- ---- ---- 2.5 µL
3 Carefully remove and discard the supernatant.
TOTAL volume 25 µL 25 µL 26 µL 25 µL
4 Resuspend the cell pellet with 50 µL Lysis Buffer by vortexing.
*We recommend that this control is prepared for each test sample.
5 Incubate the resuspended cell pellet at 37°C for 15 minutes to lyse the cells and degrade the
Store the remaining extract for each test sample at -80°C in the event further testing is
proteins. needed.
6 Heat the samples at 95°C for 10 minutes to inactivate the protease. 3 Mix gently by pipetting the reaction mixes up and down a few times. Cap tubes and centrifuge
7 Spin down cell debris at 13,000 rpm for 5 minutes at 4°C. Transfer supernatant to a new briefly to bring fluid to the bottom of the tube.
microcentrifuge tube. Do NOT use the tubes provided with the kit as these are needed for
remaining kit assays. PCR Amplification Procedure
8 Samples are now ready for PCR. If desired, these extracts may be stored at -80°C for up to six 4 Place the tubes in a thermal cycler.
months.
5 Use the following parameters for PCR:
PCR Preparation Protocol Step 1 Initial Denaturation: 94°C for 1.5 min
Precautions for PCR: This kit detects femtogram [(fg) = 10-9 µg] quantities of target DNA. Sample
Step 2 Touchdown PCR Parameters:
preparation, amplification and detection should occur in separate areas and use dedicated equipment. If
Temperature Time (seconds) Cycles
possible, assemble PCR reactions in a dedicated PCR work station with laminar flow or in a laminar flow
°C
hood. It is very important that the positive control does not contaminate other samples. Keep reactions
and components capped as much as possible. At a minimum, use pipette tips with hydrophobic filters to Denaturation 94 30
avoid cross-contamination with DNA. Annealing 70 g 60.5* 30 20

Kit Components: Thaw Universal PCR Mix, Universal Primers and Positive Control. Briefly, vortex and spin Elongation 72 45
down components to collect contents at the bottom of the tube prior to opening. *Temperature decreases 0.5°C per cycle (e.g., 70°C for 1 cycle, 69.5°C for
1 cycle, etc., to 60.5°C for 1 cycle).
Step Reaction Setup
Step 3 Continue cycling at a constant Annealing Temp.:
1 Prepare a PCR + Primers Mix by combining Universal PCR Mix with Universal Primers:
Denaturation 94 30
Component Vol. per Vol. for 5 Vol. for 10 Vol. for 20 Vol. for 40
Annealing 60 30 12
Assay Assays Assays Assays Assays
Elongation 72 45
Universal PCR Mix 20 µL 100 µL 200 µL 400 µL 800 µL
Step 4 Final Elongation: 72°C for 4 min
Universal Primers 2.5 µL 12.5 µL 25 µL 50 µL 100 µL
4°C on HOLD
TOTAL Volume 22.5 µL 112.5 µL 225 µL 450 µL 900 µL

Note on number of assays to prepare: The PCR + primers mix is needed for positive and nega-
tive controls (2 assays). We also suggest that a positive control + test sample assay is prepared
for each sample to confirm that the cell lysate (sample) does not inhibit PCR. It is recommend-
ed that test samples are prepared in duplicate.

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