Amplification of the No Template Control Due to Reagent Contamination

If you get an amplification product in your no template control (NTC), you may have one of these problems:

  1. Contamination of your reactions by DNA

    Reaction contamination can be separated into two types (select one):

  2. Primer dimer formation (SYBR® Green chemistry only)

Random contamination

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 CT values; see figure below.

NTC contamination

Solution:

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

Reagent contamination

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 AmpEraseÒ Uracil N-Glycosylase (UNG) or Uracil-DNA Glycosylase (UDG) step prior to PCR to reduce carryover contamination.
  • For optimal PCR results, Applied Biosystems recommends that you use separate working areas for PCR mix preparation, template addition, and performing the PCR reactions.

Primer dimer formation

When 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.