Frequently Asked Questions


Download: FAQ for the related products can also be downloaded as PDF file.

MiQuant® Residual DNA dPCR

Is the target sequence specific to HEK-293 cells or does it recognize any human cell?

The target sequence is specific to HEK-293 cells. Short multicopy targets in the HEK-293 genome (repeated element with ~1 million copies per genome) are amplified in this dPCR assay. The specificity has been tested as part of the validation study and a cross-reactivity with Homo sapiens was observed. This cross-reactivity was expected considering the high similarity between Homo sapiens and HEK-293 cells.

ZellShield®

Does ZellShield® contain some aminoglycosides?

The following ingredients are included in the product ZellShield®: Macrolide and polyene, lincosamide and fluoroquinolones.

What is the difference between ZellShield® and Mynox® and Mynox® Gold reagents? We have a cell culture infected with mycoplasma and would like to know which product best treats mycoplasma-contaminated cells?

ZellShield® cannot be used for curing an already contaminated cell line. You need to treat your cells with Mynox® or Mynox® Gold. If your cells are clean or you harvest cells from natural sources with a high risk of contamination (environment, students media), ZellShield® should be added to the medium to prevent mycoplasma growth.

Do I have to expect cytotoxic effects when I use ZellShield®?

No cytotoxic effects have been reported on various cell lines so far, since the product is used according to the instructions. Internal viability tests with a Vero cell line show that ZellShield® has no cytotoxic effects on the cell proliferation and cells still have very good viability. All data are summarized in a Technical Note, which can be downloaded from the product webpage.

Mynox® and Mynox® Gold

Why do we note "storage at 4 - 8 °C" instead of room temperature, when shipping is performed at room temperature?

It is absolutely practicable to ship Mynox® at room temperature for the following reasons: Mynox® is sterilized by autoclaving at 125 °C for 20 minutes and is stable at room temperature for at least 6 months even at higher temperatures. Anyhow the shelf life time of the product is much longer and sometimes customers use products even after the shelf life time has expired. To provide a product in good condition even for these circumstances we recommend long term storage as indicated on the label at +2 °C to +8 °C (Mynox® is stable at these temperatures for at least 18 months). On the attached quality certificate, you can find the information that shipment is regularly performed at room temperature to reduce additional costs for the end customer.

Mycoplasma is still detectable after treatment of cells with Mynox®.

Following the Mynox® treatment cells should be examined for remaining mycoplasma contamination after 4 passages when a sufficiently high cell density exists. Mynox® lyses the mycoplasma as an eradication mechanism. Thus, free mycoplasma DNA remains in the supernatant after treatment. With continuous cultivation and adequate cell density extracellular DNAses will hydrolyze free DNA.
Different factors might interfere with the efficiency of Mynox®. A crucial factor is the FCS concentration. FCS contains cholesterol and other target molecules for Mynox®. Hence, it is pre-requisite to avoid higher FCS concentrations in the media than suggested (final concentration of FCS must be 5 %). Another crucial aspect for efficient mycoplasma elimination is the cell concentration. If elimination of mycoplasma did not occur with the first treatment we suggest to lower the cell concentration and/or to increase the incubation time with Mynox®.
Prior to the treatment with Mynox® make sure that you don’t have cell clumps in your suspension. Extended trypsination will help to avoid the formation of cell clumps clearly. In case of adherent cells, it is highly recommended to use Petri dishes for the treatment. This will ensure that the cell suspension is not exposed to aerosols which could be produced when pipetting of the the cell suspension onto the Mynox® suspension is performed. The aerosols could stick to the surface of the vessel not being exposed to Mynox®. These contaminated aerosols could re-contaminate the cell culture later on. Therefore, it is very important to transfer the cells directly into the Mynox® suspension and not vice versa. If the mycoplasma titer at the beginning of a treatment is extremely high it might be necessary to treat the cells a second time with Mynox®. In that case it is important to provide enough time for recovery (two days/check with the microscope) to the cells after the first treatment.

When can I be certain that mycoplasmas are permanently eliminated?

You can detect mycoplasma at an early stage with the highly sensitive Venor®GeM Mycoplasma Detection Kit to exclude persisting contamination. If a few mycoplasma particles survive the treatment with Mynox®, they will grow to detectable titers within four passages.

Do I have to remove standard antibiotics when treating with Mynox®?

No, standard antibiotics like Penicillin/Streptomycin can be maintained during the treatment with Mynox®. As a basic principle, we do not recommend routine use of antibiotics. Pen/Strep mainly affects germs of the mouth and facial cavity and was introduced at times when laboratory staff used to perform mouth-pipetting. Antibiotics can affect the cellular metabolism and thus the results of the experiments (compare with: Kuhlmann, Cytotechnology 19:95-105,1996 „The prophylactic use of antibiotics in cell culture“). Bacterial contaminations can interfere latently and remain unrecognized. With Onar® Bacteria, Minerva Biolabs provides a sensitive PCR Detection Kit for bacterial contamination (Cat.-No. 12-1025).

Mynox® was ineffective in eliminating mycoplasma from virus suspensions.

The cell suspension must be free of cellular debris before treatment. Before the supernatant can be effectively treated, cellular debris should be eliminated by centrifugation (1.000 rpm, 5 min) and the pellet discarded. Such debris as well as cell membrane fragments will competitively bind Mynox®, thus decreasing the effective concentration of the elimination reagent.

At which confluence % can cell cultures be treated with Mynox® Gold?

For a successful treatment, the preparation of single cells is a necessity. We usually recommend to start the elimination procedure at 40 to 60 % confluence. In these conditions, single cells can be obtained more easily.

Mynox® was harmful to cells during treatment.

Most cases in which Mynox® was believed to be detrimental to cells, the mixture as prescribed in the Instruction Manual was not properly followed, thus resulting in a cytotoxic Mynox® concentration. Cells should be observed frequently during treatment. If cytotoxic effects are clearly evident, the treatment should be immediately stopped by medium change. For cells known to be sensitive to Mynox®, the treatment time should be reduced by passage of cells after 30 minutes for adherent cell lines and 15 minutes for suspension cell lines.

Can the cellular concentration be increased for treatment with Mynox®?

The cell concentration may be increased by 10-fold, however an overall decrease in the elimination efficiency of Mynox® should subsequently be expected.

Can Mynox® eliminate intracellular contaminants?

Mynox® does not integrate into the cellular membrane. Therefore, it cannot eliminate intracellular contamination. However, mycoplasma are extracellular contaminates. Only field isolates of mycoplasma are described as invasive, but lose this feature within a few generations at lab conditions.

Is Mynox® effective against bacteria, fungi, or chlamydia?

No, Mynox® is only effective against mycoplasma. Mynox® is especially useful against mycoplasma contamination in chlamydia cultures, as standard antibiotic treatments are damaging to chlamydia in cell lines.

Can primary cells be treated with Mynox®?

Yes, primary cells can be treated with Mynox®. However, we recommend a 10-fold increase in the cell concentration. (Note that an overall decrease in the elimination efficiency of Mynox® should subsequently be expected).

Can trypsin be a possible source of contamination in cell cultures?

Trypsin is derived from swine sources and believed to be a source of Mycoplasma hyorhinis contamination. However, Mycoplasma hyorhinis is lysed at room temperatures by trypsin within minutes. Thus it presents no source of contamination.

Can mycoplasma contamination be visible to the naked eye?

No, mycoplasma can only be observed by electron microscopy. For highly sensitive detection of mycoplasma contamination, we recommend the Venor®GeM Mycoplasma Detection Kit.

Proteinase K

How many mg of proteinase are provided?

Each vial of Proteinase K (Cat No 56-0002) contains 11 mg enzyme at ca. 30U/mg, yielding a concentration of 20 mg/ml after resuspension in 550 µl of the included rehydration buffer.

What incubation conditions do you recommend? The DNA extraction kit (Venor®GeM Sample Preparation Kit) recommends adding proteinase K then continue with the extraction (10 min, 70 °C incubation). Wouldn’t this not give the enzyme enough time to work and possibly degrade it?

The incubation with Proteinase K in our (extensively) tested protocol is performed simultaneously with the sample lysis (the step you mention at 70 °C for 10 min). The idea is to combine lysis and Proteinase K treatment in a single step, so to keep the protocol as short and yet as efficient as possible. Our data (and quality control) show that such treatment is very effective with relatively complex matrices, suggesting negligible denaturation with short incubation times (and enzyme excess!). With much more complex matrices like tissues, for instance, we would recommend to separate extraction/lysis step and Proteinase K treatment, and perform the latter at the optimum temperature for the enzyme 50-56 °C..

I have a problem with the isolation of Mycoplasma DNA with Venor®GeM Sample Preparation Kit. Sometimes there is problem with the columns clogging up due to the specific nature of the sample. Sometimes white flakes (probably of organic origin) appear in the sample and remain in supernatant after centrifugation, thereby clogging the columns.

Clogging of the columns is sometimes observed with samples containing higher amounts of proteins (e.g. vaccines, high serum concentrations..). Even if proteins may not be the main component, they could be involved in the formation of complexes between proteins and other ingredients. We recommend to try a Proteinase K treatment according to the description of the manual of the Venor®GeM sample preparation kit (optional step prior to the 70 °C incubation step).

Venor®GeM Sample Preparation Kit

I have a problem with the isolation of Mycoplasma DNA with Venor®GeM Sample Preparation Kit. Sometimes there is problem with the columns clogging up due to the specific nature of the sample. Sometimes white flakes (probably of organic origin) appear in the sample and remain in supernatant after centrifugation, thereby clogging the columns.

Clogging of the columns is sometimes observed with samples containing higher amounts of proteins (e.g. vaccines, high serum concentrations..). Even if proteins may not be the main component, they could be involved in the formation of complexes between proteins and other ingredients. We recommend to try a Proteinase K treatment according to the description of the manual of the Venor®GeM Sample Preparation kit (optional step prior to the 70 °C incubation step).

We will perform mycoplasma testing for EP-compliance. Therefore, we plan to extract mycoplasma DNA from cell supernatants with Venor®GeM Sample Preparation kit. However, we need to freeze some supernatants at -20 ºC. What do you recommend: extracting DNA from all the samples and freeze them or treat the supernatant at 95 ºC (stabilization step) and freeze them? For how long we can keep them frozen?

We strongly recommend to heat-inactivate the samples directly after sampling. If you go for freezing instead, DNases will be active during the thawing phase degrading low copy numbers of mycoplasmal DNA. After heat-inactivation, you are save and could even store the samples for one week at 2-8 °C and handle them without restrictions at RT. Both versions are equally useful: heat-inactivation or DNA extraction, both immediately after taking the sample.
With both versions you can store the samples at < -18 °C for at least 1 year.

Venor®GeM PCR/qPCR Product Line

How many Mycoplasma species can be detected with the kit?

Based on primer-genome sequence alignments, our kits can detect at least 110 species belonging to the Mollicutes class (Acholeplasma, Ureaplasma, Spiroplasma, Mycoplasma) of about 165 species which are mentioned in the literature, so far. For many – less common – species, however, there is no reliable data or sequence information. Thus, we cannot make any statement about the kit specificity for these species.
Importantly, we can detect all species mentioned in publications as significant cell culture contaminants and many others.

What is the function of the Internal Control DNA (or Internal Amplification Control)?

The Internal Control represents a second amplification reaction taking place in parallel to the target amplification (mycoplasma) in the same PCR reaction tube. It serves as a homologous control for every PCR reactions performance. The appearance of the internal control amplicon indicates that the conditions are PCR-permissive (no inhibition of the PCR). The Internal Control DNA is therefore a useful validation tool that helps ruling out false negatives.

Some samples show a strong band for mycoplasma and no band for the Internal Control (conventional PCR) / Some samples show a signal for mycoplasma in the FAMTM channel and no signal for internal control in the HEXTM channel (qPCR) Is my sample contaminated or is my PCR/qPCR run invalid?

The appearance of the Internal Control band/signal is required for negative samples only to rule out false negatives due to PCR inhibition. When your samples are strongly contaminated with mycoplasma, its DNA will be proportionally amplified. Under these conditions, the Internal Control fades out due to reagents competition. A missing internal control is therefore a perfectly normal scenario in case of heavily contaminated samples or very efficient PCR amplifications. Consequently, the results of the Internal Control become irrelevant for positive PCR reactions. Therefore, you can conclude that your PCR run is valid and unfortunately your samples heavily contaminated with mycoplasma.

I would like to freeze the supernatant after the inactivation step at 95°C and use it later on. At which temperature and for how long can I store such samples?

Based on our experience, samples can be safely stored at -20°C for at least 1 year. One can also store the inactivated supernatants at RT for 5 days or at +2- +8 °C for 14 days.

Our assays with Venor®GeM qEP have always worked well. However, this time the positive control has failed. The kit was stored correctly as advised in the protocol after rehydration etc. What could have gone wrong? Which recommendations do you have to ensure a good performance of the positive control?

The Positive Control is provided as one vial. After rehydration, this reagent is stable for frequent thawing/freezing cycles as long as you are using DNase-free pipet tips and working in a clean working area.
However, aliquoting the rehydrated Positive Control might help.
We recommend to aliquot into PCR tubes as they are usually clean, DNase-free and low-binding.

Could you please let me know if culturing the cells 48 h without antibiotics is enough before testing for mycoplasma using a Venor®GeM Mycoplasma Detection Kit?

If your antibiotics do not display activity against mycoplasma, the test can be performed without any prior antibiotics-free culture and will provide reliable information about mycoplasma contamination.
In case of mycoplasma-active antibiotics, we know that minute amounts of most mycoplasma species will grow up to easily detectable levels within 5 days of antibiotics-free culture. Fast growing mycoplasma species might be detectable after 48 h. In summary, waiting 5 days for the culture to grow in absence of such antibiotics will give you reliable results. Please keep in mind that PCR is the most sensitive method. Using other techniques, you may need to culture and delay testing even longer.

The manual of Venor®GeM Classic says that the use of Penicillin and/or Streptomycin in the culture does not affect the test. However, we use an antibiotic-antimycotic solution, which apart from Penicillin and Streptomycin contains Amphotericin B. Should we culture our cells in the absence of such antibiotic-antimycotic solution before performing the mycoplasma test?

Amphotericin B is not active against mycoplasma and should not affect the mycoplasma test.
Cultures supplemented with antibiotic-antimycotic solution can be directly tested with the Venor®GeM Classic kit.

We use Gentamycin in our media and I was wondering if that can interfere with the test. You state that penicillin and streptomycin do not significantly alter the sensitivity of the test. Will Gentamycin interfere?

Whereas the PCR assay itself will not be affected by gentamicin, the ability of mycoplasma to grow in your sample obviously will. Gentamicin is active against mycoplasma and unfortunately leads to the development of resistant strains rather quickly. Therefore, we generally recommend to test cultures, if possible, after cultivation for 1 week without antibiotic additives.

Applies only to Venor®GeM Classic
My PCR performed according to the protocol did not detect any bands (neither positive control nor internal DNA control).
The reagents were all freshly resuspended and not repeatedly frozen and thawed. I used a Hot-Start Taq Polymerase, which performed well in other PCR reactions.
What went wrong?

For our Venor®GeM Classic,we recommend our Hot-Start DNA Taq polymerase.
Using a polymerase which performs well in other PCR setups does not necessarily guarantee the success of this particular PCR assay.
The buffer provided with the kit is essential for the primers to work properly.
Not all polymerases are performing well with the kit buffer and conditions.
Getting no amplification at all usually indicates the incompatibility of the enzyme with the kit.

Applies only to Venor®GeM Classic and OneStep (conventional PCR assays)
We used Venor®GeM Classic with the Taq Polymerase from Minerva Biolabs, as suggested in the product manual. We could not detect any band in the positive or negative control lanes (the internal control is not amplified). What could be the reason?

-Among the possible causes of a PCR reaction failure, we recommend considering the possibility that the PCR cycler in-use might have a technical problem. This is not a remote possibility. In order to evaluate the performance of the cycler, we suggest testing another cycler with the same PCR set-up or another PCR set-up with the same cycler.
-Rehydration of the positive control DNA was not correct ( e.g. volume)
-PC was correctly rehydrated but too many freeze/thaw cycles were performed or DNA degradation due to contamination with DNase occurred.

Applies only to Venor®GeM qOneStep and qEP (qPCR assays)
I need advice on which qPCR-based kit for Mycoplasma detection best fits my purposes. I would assume that labeled probes and primer pairs included in Venor®GeM qOneStep are identical to Venor®GeM qEP. Is it correct? If so, which differences are there between the two kits?

Venor®GeM qOneStep is a version of the assay for Version research use only to perform the screening of cell cultures for mycoplasma contaminations. The Internal Control for PCR performance is already included in the mix. The kit is not suitable for compliance testing according to EP 2.6.7.
Venor®GeM qEP is suitable for cell culture screening and also for quality control according to the EP 2.6.7. This product has been validated and the results of the validation study can be requested. In this case, the internal control is provided as a separate vials due to its possible use also during the DNA extraction procedure. For a quick overview of the differences between the products, please have a look at the table below.

Venor®GeM qEP Venor®GeM qOneStep
Recommended Use For direct screening of cell cultures and biologicals. For EP 2.6.7/JP compliant release testing; Applicable in research and industry Applicable in research for direct testing of cell cultures and cell culture derived biologicals.
Kit Components Primer sets, nucleotides and polymerase / Rehydration buffer / separate Internal amplification control / Positive control DNA / PCR grade water Primer sets, nucleotides, internal amplification control and polymerase / Rehydration buffer / Positive control DNA / PCR grade water
Sample Volume per PCR 10 μl for EP/JP compliant testing / or 2 μl for screening 2 μl
EP/JP Compliance Yes, after appropriate sample matrix and process validation No
Validation Validation report available on request No

Applies only to Venor®GeM qOneStep and qEP (qPCR assays)
What requirements must my device fulfill in order to use the qPCR kits?

When using our qPCR mycoplasma detection kits, make sure that the cycler can detect the channels FAM™ for the mycoplasma DNA and HEX™ for the internal control (alternatively dyes for internal control VIC™ or JOE™) and that the cycler software offers the option to turn off a passive reference dye. Activating a passive reference dye leads to poor signals or signals that cannot be analysed.

Example: The AccuSEQ™ software includes a strict requirement for ROX™ as a passive reference dye. For the use of our Venor®GeM qPCR kits, this function of the passive reference dye must be switched off.
Please also note that there are different versions of the AccuSEQ™ software for which it is not always possible to display the passive reference dye.
If you have such concerns, you should contact the device or software manufacturer directly and clarify this in advance.

It is recommended to check this in the respective manual of the device and the software. A free sample of the Venor®GeM qOneStep and Venor®GeM qEP can be requested to check the compatibility of the kits with the cycler and software combination.

Venor®GeM for regulated testing (Classic and qEP)

Are these mycoplasma detection kits certified to be GMP-compliant?

Generally, in contrast to IVDs, mycoplasma detection kits are not certified or approved by any official body or governmental institution. The GMP-compliant use of the product requires a two-step validation process:

1. Product validation by the manufacturer (e.g. Minerva Biolabs)
Comprehensive validation reports are available on request for these assays.

2. Matrix validation by the user/customer:
The impact of lab-specific setup constraints and matrices on assay sensitivity and robustness must be assessed and validated by the customer. To this purpose, we offer 10CFU™ Sensitivity Standards containing inactivated and pre-titrated (10 CFUs) mycoplasma particles for easy sample preparation, available for all EP/JP-relevant species. By simple addition of the typical sample matrix to the pre-titrated mycoplasma preparations, the customer can proceed to validate his/her own matrix and setup.
We are glad to assist you during the planning and design phase of these processes. Did you know – for example – that it is not always necessary to test all mycoplasma species listed in JP/EP compendia?

Both validation reports (1. and 2.) are filed by the customer for approval of the entire manufacturing and downstream cleaning process. We actually do have a few customers around the globe, who went through this process and use our products for approved procedures.
As manufacturer, claiming to have obtained such an approval and advertising it as product compliance would be, therefore, incorrect. Unfortunately it can happen to find such incorrect claims in a few descriptions of competing products.

Venor®GeM for research testing (OneStep and qOneStep)

What is the sensitivity of the kits?

The sensitivity for all three product versions is approximately the same and ranges between 2 to 8 genome copies per PCR depending on the mycoplasma species.

Can the products also be used for the release testing of biopharmaceuticals?

The products are for screening in research applications and should not be used for the release testing of biopharmaceuticals. Consequently, they do not come with a manufacturers product validation and sensitivity is measured in GC/PCR reaction and not in CFU/ml as required for release testing.

Do the kits require sample preparation?

For screening, these three kits do not require necessarily sample preparation. DNA extraction of the samples prior to PCR is necessary if you want to test thawed samples or cryo samples directly. High concentrations of FBS and DMSO can inhibit the PCR reaction.

Proficiency Testing Program

What does “NAT” mean?

Nucleic Acid Amplification Technology

So, does NAT mean PCR?

Not necessarily, but NAT-based tests include PCR. Any technique, which is based on the detection of DNA can be used.

Will you provide a kind of an “official certificate of external quality assurance” based on the results obtained by the user?

Yes. An official certificate for participation and/or results within the range will be issued.

Do you require that the participants declare what method of detection they use?

Yes, as this will give the participants a statistical overview on available test systems and their characteristics.

If I register via our local distributor, who will issue the certificate?

It’s the distributor’s choice to forward the contact details. For anonymous participants, the distributor will receive the test samples and will forwardthem to the participant. The results will be transferred by the distributor to Minerva Biolabs for evaluation. The certificate will be issued by the distributor. Minerva will not be aware of end customers contact information. In all other cases, Minerva Biolabs will issue the certificate.

Can users who perform testing based on colorimetric detection, ELISA or another detection method participate?

No, since the material is inactivated, colorimetric assays cannot be used. The inactivation method will irreversibly alters both the microorganism viability and its structural properties, making the samples incompatible with ELISA.

You say that you will supply a comparable set of samples. But in fact for each participant, the set will be different? Some of participants will have vials with mycoplasma; some other could have only positives or negatives …?

All participants will receive exactly the same material. Material from different proficiency tests will differ in formulation.

Do participants using an in-house method receive some kind of official certificate that their assay is ok?

No. This proficiency test is exclusively organized for Sartorius Stedim and Minerva Biolabs customers.

Are those samples enough for a costumer to compare e.g. 2 or 3 different methods?

Each sample will contain a lyophilized pellet which should be rehydrated with 1000 µl of sample medium. This volume should be enough for many PCR reactions and even in case a DNA extraction is required.

Can we compare our in-house test to a commercial test by using these sample sets? Is the price the same for using the samples for this purpose?

Of course participants can compare different methods with the same sample set. However, only the method performed with a Sartorius Stedim or Minerva Biolabs kit will be certified.