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| 1. Negative reaction became blue color after PCR reaction. Why and how to prevent? |
(1) Cross contamination between samples. When adding samples and positive control, it is very easy to contaminate the pipettes or working area. It is strongly recommended to prepare
complete-PCR-cocktail in a separate area (aliquoting-area) and use a set of designated pipettes. A PCR hood is strongly recommended as
“aliquoting-area”. Do not cough, sneeze when preparing
complete-PCR-cocktail and aliquoting, and wear a mask if possible. Add samples and positive control using another designated 10ul pipettes in another separate area (adding-sample-area). During the whole process of adding samples and positive control, the cap of negative control tube should always be closed and do NOT perform any pipetting step for the negative control tube. To minimize the cross contamination, do NOT add 1ul pure-water as negative control in “adding-sample-area”. Negative control tube should NOT be opened in “adding-sample-area”. After PCR completion, NEVER open the caps of PCR tubes to prevent the amplified products from contaminating the whole lab area.
(2) Observation of color during PCR. Never check color during PCR, never take the PCR tube out during PCR, never open the PCR lid until PCR finished. Because they significantly boosts non-specific amplification which can turn the color to blue.
(3) False positive. It happens occasionally with a rate about 8%. Repeat the reaction of negative control.
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| 2. Positive control kept purple and did not turn to blue after PCR completion. |
| The synthesized gene of mycoplasma provided for positive control is diluted. Freeze-thaw can degrade it significantly. Freeze-thaw more than 2 times may fail the Positive Control. Aliquot it into single-use tubes at the first time of thawing, and store at -20°C. The single-use aliquot can only be thawed and used for one time. NEVER re-use the single-use aliquot.
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| 3. May I run agarose gel electrophoresis of PCR product? |
| No. The 60 cycles employed by the kit is to ensure the chemical to develop the color. It also results in over-amplification which gives smears and absence of sharp band in gel electrophoresis. Therefore, run electrophoresis is not informative. In addition, opening PCR tubes gives rise to contaminate the whole lab area, which is a disaster and result in false positive for future reactions.
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4. May I freeze the
complete-PCR-cocktail?
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| No. Once prepared, the
complete-PCR-cocktail should only be stored at 4°C for 1 month. It can NOT be frozen at any time.
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5. What is the copy number of synthesized gene in component D? May I use it as a reference to estimate the titer of mycoplasma in the contaminated samples?
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| It is in the range of
103-106
copies/ul. However, the synthesized mycoplasma gene in such concentration is unstable and very susceptible to degradation upon freeze-thaw. Therefore, the actual copy number of synthesized gene in component D is uncertain. It is not practical to use it as a reference to estimate the titer of mycoplasma in contaminated samples.
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6. The
complete-PCR-cocktail is not purple but violet color. May I continue to use it for
PCR?
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| Yes, it can be used without problem. Be sure to run negative control as a color reference. After PCR completion,
samples showing blue or sky-blue color other than the violet color of negative control are mycoplasma positive. The color of
complete-PCR-cocktail is influenced by temperature. Reconstitution of
complete-PCR-cocktail on ice will give a color toward to reddish (purple color for example). Reconstitution of
complete-PCR-cocktail at room temperature will give a color toward to bluish (violet color for example).
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7. After PCR
reaction, a sample shows color between negative control and positive control. Is it mycoplasma positive or negative?
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| Samples showing sky-blue or blue are
mycoplasma-positive. Sky-blue color means high titer of mycoplasma contamination, whereas blue color means medium or low titer of
mycoplasma. Samples showing violet or purple are
mycoplasma-negative. If a sample shows color between positive and negative, 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?
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The following crude samples can be used directly as PCR template for the kit:
- cell cultures (both adherent cells and non-adherent
cells)
- frozen cell line stored in liquid
nitrogen
- culture medium
(DMEM, RPMI 1640) and serum (FBS, FCS)
- Complete culture medium containing 5%-20%
FBS/FCS
- cell freezing solution (90%
FCS, 10% DMSO)
- Penicillin/Streptomycin (100X stock
solution)
- Non-essential Amino Acid (100X stock
solution)
- 1xPBS
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The following samples are not compatible to the kit:
- Trypsin/EDTA
solution
- Trypsinized
cells
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