How do I know there was an issue with a qPCR run?
- Negative Control Failed
- Positive Control Failed
- No Target Amplification & Positive Control Failed
My positive control failed; what to check first?
- Confirm the positive control is being stored correctly
- Confirm the positive control was rehydrated correctly, incubating for at least 5 minutes and occasionally mixed by swirling the tube
- Confirm you are using a fresh vial of positive control. It is best to avoid multiple freeze-thaw cycles
How can I avoid common issues?
The best practice is to have distance between work areas for each step in the process to prevent contamination. The steps include sample preparation & preservation, DNA purification & q-PCR analysis.
However, if space is limited, the best defense against contamination is proper cleaning protocols (10% bleach followed by 70% ethanol), frequently changing pipet tips and gloves, and keeping reagent bottles or sample tubes closed if they are not being actively used.
Please be sure to use care in handling the positive control (use fresh pipet tips and cap tubes as soon as the Nuclease-Free Water is added). The positive control DNA is highly concentrated, so care must be taken not to contaminate other samples with the Positive Control DNA to prevent inaccurate results.
Beyond positive control contamination, there can also be cross-sample contamination which can be avoided using the same practices (switching pipet tips frequently, proper surface cleaning, keeping bottles/tubes closed, etc.)
How do I know if I have inhibition?
- No target amplification when I expected to identify a target
- My internal control failed
What is internal control?
Internal Controls are intended to confirm that there are no inhibitors present in the reaction and that the master mix amplifies properly to validate a qPCR result. Failed internal controls may indicate the presence of inhibitors.
Which corrosion targets have internal controls?
The following corrosion panel assays have internal controls:
- Total Bacteria
- Total Archaea
- Corrosive Methanogens
LuminUltra offers eight corrosion-related assays to identify specific microbes of concern, new users may choose to include one of the qPCR targets: Total Bacteria, Total Archaea, and Corrosive Methanogens assays, which include an internal control to identify inhibition.
If analyzing qPCR results that may have inhibition, a 1:10 dilution of the eluted DNA may be prepared to dilute out qPCR inhibitors. Note any dilution performed on the sample for later analysis.
DNA purification and qPCR assay setup require good pipettor techniques to ensure accurate liquid volumes are used throughout the protocol to get the best results. Learn more about Best practices for qPCR pipetting here: Quick Guide to qPCR Pipetting
qPCR Strip Tube Prep
Please make sure all the liquid is at the bottom of the qPCR strip tubes before placing them in the thermal cycler. To do this, pick up the strip tubes and use a robust downward motion to ensure the contents of the qPCR tubes are at the bottom of the tube.
Preserve & Purify Standard
The wash buffer contains Ethanol, which, if left over in the sample, can inhibit qPCR and other downstream protocols. Ethanol is a strong PCR reaction inhibitor; small amounts can interfere with sample results. Here are some best practices to avoid ethanol inhibition: remove as much as possible. If Magnetic Beads are aspirated, gently pipette the liquid back into the inside of the cap to let the beads attract to the magnetic and repeat the wash buffer removal. Please allow enough time for the beads to dry before the elution step.