PCR Decontamination Kit

The PCR Decontamination Kit removes contaminating DNA in PCR master mixes, without reduction of PCR sensitivity.

  • The double-strand specific property of the dsDNase allows decontamination with primers and probe present.
  • Efficient for end-point PCR and probe-based qPCR.
  • Contaminating bacterial DNA is reduced to levels below detection limit.
  • Fast and easy protocol.

Decontamination of master mixes without reduction of sensitivity has always been a challenge. Especially when minor amounts of DNA are targeted, contaminating DNA is a major problem. Any loss of sensitivity in the qPCR assay caused by the decontamination protocol is unacceptable.

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Protocol

  1. Mix primers and probes with the master mix and kit content
  2. Incubate 20 minutes at 37ËšC
  3. Inactivate 20 minutes at 60ËšC
  4. Add template and run qPCR

dsDNase is irreversibly inactivated at 60ËšC in presence of DTT, ensuring that any template added after inactivation remains safe from digestion.

Protocol for removing contaminating DNA from 20 µl reactions but can be scaled up or down by adjusting the volume of the kit contents.

Image

Figure 1: PCR Decontamination Kit protocol

Figure 2: Untreated and decontaminated qPCR 2x master mix was used for analysis of an E. coli gDNA 10-fold serial dilution with 5 steps. NTC samples were included and all plots of the serial dilution show an average of three replicates.

Treatment with the PCR Decontamination Kit does not reduce the sensitivity for the PCR. Even when diluting the DNA, the Cq-levels were not significantly altered (Figure 2).

Kit Contents

  • DTT (Inactivation Aid)
  • dsDNase (100 reactions)

Storage Conditions: Store at -20ËšC.
Sample Material:The kit can be used to reduce contaminating DNA in both ordinary PCRs and probe based qPCR mixes. Also efficient for some SYBR based qPCR mixes.
Quality Control: The kit is tested for absence of RNase.

Publications

PCR Decontamination Kit Applications

  1. The Not-so-Sterile Womb: Evidence That the Human Fetus Is Exposed to Bacteria Prior to Birth.
    Stinson LF, Byce MC, Payne MS, Keelan JA.
    Front. Microbiol. 2019; 10: 1124.

  2. Characterization of the bacterial microbiome in first-pass meconium using propidium monoazide (PMA) to exclude nonviable bacterial DNA.
    Stinson LF, Keelan JA, Payne MS.
    Lett Appl Microbiol. 2019; 68(5):378-385.

  3. Identification and removal of contaminating microbial DNA from PCR reagents: impact on low-biomass microbiome analyses.
    Stinson LF, Keelan JA, Payne MS.
    Lett Appl Microbiol. 2019; 68(1):2-8.

  4. Multiplex detection of extensively drug resistant tuberculosis using binary deoxyribozyme sensors.
    Bengtson HN, Homolka S, Niemann S, Reis AJ, da Silva PE, Gerasimova YV, Kolpashchikov DM. Rohde KH.
    Biosens Bioelectron. 2017; 94, 176-183.

  5. Microbial Typing by Machine Learned DNA Melt Signatures. 
    Andini N, Wang B, Athamanolap P, Hardick J, Masek BJ, Thair S, Hu A, Avornu G, Peterson S, Cogill S, Rothman RE, Carroll KC, Gaydos CA, Wang JT, Batzoglou S, Yang S.
    Sci Rep. 2017; 7:  42097.