Amplification of the No Template Control (NTC)

In this situation, NTC contamination randomly occurs when loading the DNA templates into the PCR plate. If the contamination is due to plate loading, the NTC will show amplification in some or all of the NTCs at varying C_T values; see figure below.

NTC contamination

Solution:

• Use clean working practices to avoid template contamination.
• To avoid contamination from previous PCRs, incorporate an Applied Biosystems™ AmpErase™ Uracil N-Glycosylase (UNG) or Uracil-DNA Glycosylase (UDG) step prior to PCR to reduce carryover contamination.
• For optimal PCR results, we recommend that you use separate working areas for PCR mix preparation, template addition, and performing the PCR reactions.

One or more reagents are contaminated. If this is the case, amplification of the NTC replicates should be close, as shown below, because the same amount of template was loaded onto the PCR plate.

Amplification as a result of reagent contamination

All reagents used in real-time PCR reactions (i.e., master mix, water, and primers/probes) can become contaminated with template. This can be due to a lack of GLP (good laboratory practice) resulting in random template contaminations. In addition, if you perform pre-PCR and post-PCR procedures in the same area, the PCR product carryover from a previous reaction can contaminate a new PCR plate and cause positive results.

Solution:

• Use clean working practices to avoid template contamination.
• To avoid contamination from previous PCRs, incorporate an AmpErase Uracil N-Glycosylase (UNG) or Uracil-DNA Glycosylase (UDG) step prior to PCR to reduce carryover contamination.
• For optimal PCR results, we recommend that you use separate working areas for PCR mix preparation, template addition, and performing the PCR reactions.

When Applied Biosystems™ SYBR™ Green dye is used in real-time PCR, any primer dimers will cause higher background, and may lead to a generation of CT<40 for NTC samples. The primer dimer is not usually detected unless a dissociation profile is generated. Because the background is higher, large amounts of primer dimers may alter the CT of your experimental samples and change the expression level interpretation.

		A SYBR Green amplification plot prior to the dissociation curve. In this view the presence of primer dimers cannot be determined.
		A dissociation curve following a SYBR Green real-time reaction. The presence of primer dimers can be easily identified in the dissociation curve by the additional peak(s). The primer dimer peaks are usually seen at low melting temperatures.

Solution:

1. Optimize primer concentration using the following combinations:
   

   Reverse primer (nM)	Forward primer (nM)
   	100	200	400
   100	100/100	200/100	400/100
   200	100/200	200/200	400/200
   400	100/400	200/400	400/400

2. Use an amount of primer that produces no primer dimer and gives optimal amplification efficiency.