PROBLEM |
PROBABLE CAUSE(S) |
SOLUTION(S) |
qPCR traces show low or no amplification |
Reagent omitted from qPCR assay
Reagent added improperly to qPCR assay |
- Verify all steps of the protocol were followed correctly
|
Incorrect cycling protocol |
- Refer to the proper qPCR cycling protocol in product manual
|
Incorrect channel selected for the qPCR thermal cycler |
- Verify correct optical settings on the qPCR instrument
|
DNA template or reagents are contaminated or degraded |
- Confirm the expiration dates of the kit reagents
- Verify proper storage conditions provided in this user manual
- Rerun the qPCR assay with fresh reagents
- Confirm template input amount
|
Inconsistent qPCR traces for triplicate data |
Improper pipetting during qPCR assay set-up |
- Ensure proper pipetting techniques
|
qPCR plate film has lost its seal, causing evaporation in the well. The resulting qPCR trace may show significantly different fluorescence values relative to its replicates |
- Ensure the qPCR plate is properly sealed before inserting into the qPCR thermal cycler.
- Exclude problematic trace(s) from data analysis.
|
Poor mixing of reagents during qPCR set-up |
- Make sure all reagents are properly mixed after thawing them
|
Bubbles cause an abnormal qPCR trace |
- Avoid bubbles in the qPCR plate
- Centrifuge the qPCR plate prior to running it in the thermal cycler
- Exclude problematic trace(s) from data analysis
|
DNA standard curve has a poor correlation coefficient/ efficiency of the DNA standard curve falls outside the 90–110% range |
Presence of outlying qPCR traces |
- Omit data produced by qPCR traces that are clearly outliers caused by bubbles, plate sealing issues, or other experimental problems
|
Improper pipetting during qPCR assay set-up |
- Ensure that proper pipetting techniques are used
|
Reaction conditions are incorrect |
- Verify that all steps of the protocol were followed correctly
|
Bubbles cause an abnormal qPCR trace |
- Avoid bubbles in the qPCR plate
- Centrifuge the qPCR plate prior to running it in the thermal cycler
|
Poor mixing of reagents |
- After thawing, make sure all reagents are properly mixed
|
Threshold is improperly set for the qPCR traces |
- Ensure the threshold is set in the exponential region of qPCR traces
- Refer to the real-time instrument user manual to manually set an appropriate threshold
|
Melt curve shows different peaks for low input samples |
Non-template amplification is occurring
Infrequently, denaturation of a single species can occur in a biphasic manner, resulting in two peaks |
- Compare melt curve of NTC to samples
- Redesign primers with a Tm of 60°C or use our Tm calculator to determine the optimal annealing temperature of the primers
- Perform a primer matrix analysis to determine optimal primer concentrations
|
No template control qPCR trace shows amplification, NTC Cq is close to or overlapping lower copy standards |
Reagents are contaminated with carried-over products of previous qPCR (melt curve of NTC matches melt curve of higher input standards) |
- Replace all stocks and reagents
- Clean equipment and setup area with a 10% chlorine bleach
- Consider use of 0.2 U/μl Antarctic Thermolabile UDG to eliminate carryover products
|
Primers produce non-specific amplification
(melt curve of NTC does not match melt curve of higher input standards) |
- Redesign primers with a Tm of 60°C or use qPCR primer design software
|
