PCR Mycoplasma Detection Kit
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PCR Mycoplasma Detection Kit
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Cat.No.: G238
Quantity: 100 Reactions
Price: $165.00
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Data Sheet
Description
  Mycoplasma detection is often difficult due to its lack of visable appearance, therefore it can be afflicting your valuable cell 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 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.
Features
-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.
Product Brochure  
Detectable Mycoplasma Species/Strains List  
Notes
1. Mycoplasma contamination detection and mycoplasma elimination customer service are also provided, please refer to the Mycoplasma Detection and Elimination Service page for details.
Product Documents
Product References
FAQs
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What is the protocol for freezing iPSCs?
1) 4-5 days after cell split, when iPSCs reach a confluency of 30-60%, aspirate out spend medium and add 1ml 0.5mM EDTA/PBS. Incubate cells at 37C for ~5min.
2) When colonies start to detach, gently harvest all the cells into a 5-ml or 15-ml or 50-ml tube by pippeting 1-2 times. Avoid multiple pippetting. Breaking down clumps into single cells may substantially decrease cell survival. May add iPSC medium to harvest all the cells.
3) Spin down at 200g for 2-3 minutes.
4) Carefully aspirate out supernatent and gently resuspend cells in 0.5ml iPSC medium.
5) Add 0.5ml freezing medium (TM023) and ROCK inhibitor. Mix well by flipping the vial and transfer the medium to a cryovial.
6) Finally transfer the vial to -80C freezer for short-term storage (days to weeks). After cells are frozen, you may then transfer the vial to liquid nitrogen tank for long-term storage (years).
What is the recommended seeding density for iPSC?
IPSCs require seeder cells before thawing and plating the IPSC. Seed ~3x10^5 feeder cells in each well of a 6-well plate. After thawing the iPSCs, put all the cells from the vial into 1 well. After several days, you may start splitting the iPSCs into multiple wells depending on how many cells survived.

The density mentioned above is for the seeder cells which should be at 3x10^5. Afterwards, please plate the entire IPSC into one well at 10^6 concentration.
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 positive control in this kit as insurance for a positive PCR reaction.
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 G238 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 +4C 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 4C 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.
When I run the PCR product on Agarose gel, my negative control is clean, my positive control shows a 500 bp band, but my sample shows 550 bp. Does this band fall within the expected results?
The length of the PCR product will vary between 370-550 bp depending on the different mycoplasma species/strains. The positive control PCR product will be ~450bp.