Bio-Rad SsoAdvanced™ Universal SYBR® Green Supermix - Manual (Page 25)

| 19 SsoAdvanced ™ Universal SYBR ® Green Supermix Instruction Manual | 19
Control Samples/Wells Are Not Performing as Expected
If your non-template control (NTC) wells indicate amplification, you need to determine the
source. If primer dimers are not the cause (please review the prior section), then the most likely
cause is nucleic acid contamination. This can result from the following, but is not limited to:
■ ■
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 non-filtered pipet tips
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.
If your no-RT control wells indicate amplification, you need to determine the amount
of gDNA contamination present in your cDNA sample(s) to understand the impact on
your data.
Fig. 17. Serial dilution of template where the
lowest dilution point (100 pg) has lower Cq values
than expected due to primer dimer amplification.
Initial DNA
Cq
10 ng
100 ng
100 pg
1 ng
1 µg