Does the cell culture need to be confluent before collecting samples?
No. Due to the high sensitivity of the EZ-PCR™ Mycoplasma Detection Kit, it is not necessary to culture the cells to confluency for an accurate readout.
What happens if samples are collected for mycoplasma testing more than 48 hours after the last media exchange?
If cell culture media is older than 48 hours when collected, cellular byproducts can accumulate that can inhibit the PCR reaction. In this case, it is best to replace media and collect for PCR between 24 and 48 hours later. If a sample collected after the 48-hour timeframe must be used, DNA extraction is recommended prior to PCR amplification.
A good indication that the samples were collected after more than 48 hours is either inhibition of the Internal Control, or a "smear" across the sample lane due to DNA debris.
How can I be sure that a faint positive band means my sample is mycoplasma positive?
To know for sure, a sample producing a faint positive band should be re-tested. If a faint band persists in the subsequent assay, the sample should be considered mycoplasma positive. If the faint band no longer appears in the subsequent assay, the sample is negative.
How definitive are the given gel electrophoresis parameters?
Running the 2% agarose gel at 100 volts for 75 minutes is a recommendation but may vary depending on the apparatus. If recommendations for specific gel electrophoresis setups are needed, one option is to prepare and run a pre-made 2% E Gel agarose gel (Thermo Fisher) according to protocol with the appropriate program on the E-Gel® iBase™ Power System (Thermo Fisher) to run for 26 minutes.
Troubleshooting
The internal control band is faint or missing, but my test sample is positive (band at 270bp)
High amounts of mycoplasma within the sample can lead to PCR competition, resulting in a faint internal control band at 270bp, or no internal control band at all. Try testing a more dilute concentration of the sample.
The negative control also shows a positive band (at 357bp)
The kit reagents may have become contaminated. Use the same kit to run another test with positive control and negative control only. If the negative control still shows a positive band, check sterility practices and dispose of kit.
Bands appear lower than 100bp
Primer dimerization may have occurred. While this often does not affect the sensitivity of the test, it could be due to old reagents that have degraded over time. A new kit may be needed to ensure clear and easily interpreted results.
Bands appear “smeared”
The collected sample may contain too many cellular bi-products. Confirm that the test sample was not taken longer than 48 hours after media exchange. Try testing a sample within 24 hours of media exchange.
