Troubleshooting and FAQ

1. Negative reaction became blue color after PCR reaction. Why and how to prevent?

This can be due to a few reasons:

• Cross-contamination between samples: It is strongly recommended to prepare the complete-PCR-cocktail in a separate area with designated pipettes. A PCR hood is ideal. Do not cough or sneeze when preparing reagents. Add samples and the positive control in a different area. The negative control tube should never be opened in the sample-adding area. After PCR, NEVER open the caps of PCR tubes to prevent amplified products from contaminating the lab.
• Observation of color during PCR: Never check the color during the PCR run. Do not take tubes out or open the PCR lid until the reaction is finished, as this can boost non-specific amplification and cause a color change.
• False positive: This happens occasionally (~8% rate). Repeat the reaction for the negative control.

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2. Positive control kept purple and did not turn to blue after PCR completion.

The synthesized mycoplasma gene provided for the positive control is diluted and can degrade significantly with freeze-thaw cycles. Freeze-thawing more than twice may cause the positive control to fail. It is recommended to aliquot it into single-use tubes upon first thaw and store at -20°C. A single-use aliquot should only be thawed once. NEVER re-use it.

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3. May I run agarose gel electrophoresis of PCR product?

No. The 60 cycles employed by the kit are to ensure the chemical develops the color. This also results in over-amplification, which gives smears and an absence of a sharp band in gel electrophoresis. Therefore, running a gel is not informative. Additionally, opening PCR tubes can lead to contamination of the entire lab area, resulting in false positives in future reactions.

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4. May I freeze the complete-PCR-cocktail?

No. Once prepared, the complete-PCR-cocktail should only be stored at 4°C for up to 1 month. It CANNOT be frozen at any time.

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5. What is the copy number of the synthesized gene in component D? May I use it as a reference to estimate the titer of mycoplasma in contaminated samples?

The copy number is in the range of 10³-10⁶ copies/µl. However, the synthesized gene at this concentration is unstable and very susceptible to degradation upon freeze-thaw. Therefore, the actual copy number is uncertain, and it is not practical to use it as a reference to estimate mycoplasma titer.

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6. The complete-PCR-cocktail is not purple but violet color. May I continue to use it for PCR?

Yes, it can be used without a problem. Be sure to run a negative control as a color reference. After PCR, samples showing a blue or sky-blue color, different from the violet of the negative control, are mycoplasma positive. The color of the cocktail is influenced by temperature; reconstitution on ice tends to yield a reddish/purple color, while reconstitution at room temperature yields a bluish/violet color.

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7. After the PCR reaction, a sample shows a color between the negative and positive controls. Is it mycoplasma positive or negative?

Samples showing sky-blue or blue are mycoplasma-positive. Sky-blue indicates a high titer of contamination, whereas blue means a medium or low titer. Samples showing violet or purple are mycoplasma-negative. If a sample shows an intermediate color, repeat the reaction to confirm the result.

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8. What kinds of crude samples can be tested by the Visual-PCR Mycoplasma Detection kit?

The following crude samples can be used directly as PCR templates:

• Cell cultures (both adherent and non-adherent cells)
• Frozen cell lines stored in liquid nitrogen
• Culture medium (DMEM, RPMI 1640) and serum (FBS, FCS)
• Complete culture medium containing 5%-20% FBS/FCS
• Cell freezing solution (e.g., 90% FCS, 10% DMSO)
• Penicillin/Streptomycin (100X stock solution)
• Non-essential Amino Acid (100X stock solution)
• 1xPBS

The following samples are NOT compatible with the kit:

• Trypsin/EDTA solution
• Trypsinized cells

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