Bio-Rad SsoAdvanced™ Universal Probes Supermix - Manual (Page 25)

| 19 SsoAdvanced ™ Universal Probes Supermix Instruction Manual | 19
If you suspect the standard curve and dilution points are not within the MIQE guidelines
of 90–110% PCR efficiency with an R
2 of 0.99 or greater, consider the following
corrective actions:
1. Ensure that the standard curve covers at least 5–6 logs of dynamic range. When the
standard curve is too small, the variability of the true efficiency greatly increases.
2. If the R 2 is <0.98, review the standard curve data points for outliers. Remove any outliers
where the ∆Cq is >0.5 for the group. For example, if your 100 pg dilution point has Cq
values of 29.2, 29.6, and 30.5, you should remove the Cq value of 30.5. If there are too
many outliers, it may be a sign of other technical issues.
Control Samples/Wells Are Not Performing as Expected
If your non-template control (NTC) wells indicate amplification, you need to determine
the source. Although the most likely cause is nucleic acid contamination, other possible
causes include:
■ ■
Pipetting template into the NTC well
■ ■
Sample from adjacent wells being aerosolized while pipetting or removing the plate seal
after samples have been loaded
■ ■
Contaminated plate, water, primers, or supermix
■ ■
Use of nonfiltered pipet tips
■ ■
Degraded probe
1. Evaluate your current workflow and adjust as needed. If you suspect your reagents are
contaminated, the best method to determine the source is to replace them one at a time
starting with the water, which is a common source of contamination. Next, make a fresh
dilution of primers from the stock solution. And finally, use a new aliquot of the supermix.
Discard any identified contaminated reagent from the lab.
2. If the problem persists, evaluate the background noise for the entire real-time PCR run
across all wells. If the signal is unusually high compared to prior runs, your probe may
be degrading. When this occurs, the high temperatures cause the probe to cleave, thus
releasing the reporter dye into solution and allowing fluorescence. Probes should be
aliquoted upon receipt into amber tubes and should not be exposed to freeze/thaw
cycles > five times, as this causes premature degradation.