Mycoplasma detection is often difficult due to its lack of visible appearance, therefore it can be afflicting your valuable cells and affecting your results without your knowledge. Cells contaminated by mycoplasma species can have changes in proliferation, metabolism, gene synthesis and processing, and even adhesion properties. The solution for quick and easy mycoplasma detection is abm’s PCR Mycoplasma Detection Kit.
The PCR Mycoplasma Detection Kit by abm allows for fast and reliable identification of mycoplasma contamination in cell cultures. Mycoplasma DNA in the cell culture supernatant is amplified via PCR and visualized using gel electrophoresis. In addition to the short detection process (less than 2 hours), the easy handling and high sensitivity makes this PCR Mycoplasma Detection Kit a convenient tool for routine examination of cell cultures and media.
- Direct addition of cell culture supernatant to PCR reaction – no DNA isolation/purification steps required
- Ready-to-use primer mix – reduces variability
- Able to detect numerous mycoplasma species
- High sensitivity
- Included positive control to verify negative results
- Rapid results in 2 hours
Supplied by Applied Biological Materials (abm). Usually ships in 3 business days.
Do you need any kind of initial training to use your kit?
This kit is PCR based and very easy to use. There is no initial training for that. But we will be more than happy to answer any questions related to the use of this kit.
Do you provide or sell a positive control so that we can be sure the assay worked?
Yes, we do have a positive control included in the kit.
Does your Mycoplasma detection kit come with an internal control for each sample to ensure that the reason the sample appears negative is not because the reaction itself simply did not work?
We provide the positive control in this kit as insurance for a positive PCR reaction.
What size plate, dish or flask should I use for the culturing of my cells?
We recommend culturing your cells in a 6-well plate or 10cm dish.
What is the basis of the positive control in the mycoplasma kit?
It is DNA fragments containing proprietary sequences diluted in medium.
Can tissue culture supernatant be frozen and thawed for the test?
I noticed the product arrived with precipitation. Is that normal?
That is normal as the contents are concentrated. You may warm the product in a 37 degrees Celsius water bath until the sample has completely dissolved.
Can this kit be used for DNA samples extracted from cell pellets?
Yes, this kit will also work for extracted DNA samples
I kept the cells in the presence of gentamycin, Should I use antibiotics-free media to detect mycoplasma in the media?
It is not necessary to remove routine antibiotics (e.g. penicillin, streptomycin, and gentamicin) from the media prior to PCR detection, as they will not interfere with the assay.
What is the detection limit of your kit?
Our kit is able to detect as low as 10 copies of Mycoplasma/sample
What is the % of agarose gel that would work best for the Mycoplasma PCR kit?
You may use standard 1% agarose gel and this will be the most cost-effective option. The product size is small (less than 500 bp) so 1.5% to 2% gel can also be used.
Our cells are grown in suspension and would not achieve 80% confluence, is there a target cell density (cells/ml) we should achieve to ensure we will detect the mycoplasma?
Mycoplasma testing is more dependent on how recently the cells have been subcultured. Seeding density does not play a major role when using this kit. We recommend cells be around at least 3-5 days old in culture without changing any fresh media so as to detect secreted mycoplasma in culture supernatant (i.e. the cell culture medium should not be refreshed/changed for 48-72 hours prior to collection).
We might not have access to the ultracentrifuge until a later date. Can we can collect the media samples and freeze or store at 4°C until we centrifuge etc.?
Storing the media samples at 4°C until you are able to centrifuge should be fine.
Can I use cryogenically frozen supernatant?
Using samples stored at -80C should be fine for this kit. If you thawing out the cells and performing mycoplasma testing right away, usually when thawing the cells the supernatant from the vial is diluted when adding to the cell culture vessel. As long as the DMSO is diluted and the DMSO content is less than 0.5% final in the PCR reaction it should not cause a problem. Too much DMSO would alter the Tm of the PCR buffer and thus affect amplification.
What suggestions can you provide for the gel electrophoresis step?
We normally use 0.5X TAE buffer to cast 1% gel and also use the same buffer to run the electrophoresis in-house at 100V for about 20 minutes.
Is the positive control a mixture of more than one strain of mycoplasma DNA or inactivated mycoplasma that cannot divide?
The Mycoplasma positive control is a cloned vector containing a section of Mycoplasma gDNA sequence.
Since I have such a large number of cell lines to test, can I store the cell culture supernatant prior to use in the PCR, or does it have to be freshly collected?
In general, the more fresh the sample is when running the PCR, the better; in keeping with this, it is normally advised to wait until all cell lines are ready to go prior to collecting the supernatant for each. However, if necessary, you can certainly store collected supernatant at +4˚C storage for short-term (1-2 days) storage; note that we highly recommend avoiding repeated freeze thaw cycles.
I noticed that there are clear crystals in the product solution. Is it still functional?
The qPCR mastermix should still be functional. The crystals are from our proprietary buffering salt, which will be dissolved eventually when you warm the solution up to room temperature or even 37C. All our qPCR mastermixes are very stable and can withstand this kind of warming cycle. Once the salt is fully dissolved, the vial can be kept at 4˚C for 2-3 months.
How many days after thawing my cells can I perform this mycoplasma test?
We recommend the cells should remain in culture for at least 48-72 hours prior to screening for the presence of mycoplasmas, and that the media sample collection only be done once the cells have reached at least 80% confluence.
What size dish or flasks do you recommend the cells should be cultured in?
We encourage customers to culture their cells in either a 6-well plate or 10 cm plate.