![]() ![]() Hence, if a positive NTC is observed, testing with different master mixes will help rule out the master mix as the cause of contamination.įor research use only. Blocking oligos and/or clamps can also be used to block amplification of common sequences and enhance the amplification of a rare sequence.Īs new sequences are deposited into the NCBI database, it is essential to perform a BLAST search of every primer and probe sequence used in PCR to check for cross reactivity.īacterial ribosomal sequences can be amplified readily from virtually any bacterial source, including bacteria-derived Taq polymerases used to amplify them, as well as non sterile tubes and pipette tips. Alternatively, a conserved, species-specific gene may be used. In 16S rRNA experiments, a better approach may be to choose a unique sequence from the hypervariable region of 16S rRNA gene. Choose a hypervariable region for the species-specific gene or novel sequence For such applications, genomic DNA, rather than RNA or cDNA, is used as the sample. While not commonly used for RT-PCR or gene expression studies, rRNA sequences are often used for characterizing environmental species diversity, such as bacterial strains within the intestine or in saltwater marshes. There are some DNA sequences, such as bacterial genes for 16S or 23S rRNA, that can be found almost anywhere. Thus, it is important to consider the pervasiveness of the chosen primer or probe sequences. The exponential PCR process can amplify a single copy of DNA. Am I amplifying DNA from my reagents or consumables? This section addresses false positives that occur during bacterial research when primers and probes are designed to detect common sequences, such as ribosomal RNA (rRNA). In PCR experiments, amplification in the “no template control” (NTC) before the ~38th cycle with probe-based assays (or ~34th cycle when using intercalating dyes) is a sign of false positives and/or contamination. Use a hypervariable region for the species-specific gene or a novel sequence as a control if you are repeatedly seeing false positives.Īvoiding false positives when using universal primers for bacterial identification.Signal to noise assessment, mass spectrometry, or a fluorometric scan can be used to address possible probe degradation. In such situations, there may still be signal from free dye and/or high background. Be sure to replace all reagents and stock buffers and thoroughly clean PCR preparation areas.If you do find contamination in your NTC sample Perform melt curve analysis after PCR to check for primer-dimers. Late amplification may not be indicative of a positive NTC as it could also be a result of dimer amplification.Place the no template control (NTC) wells as far as possible from positive samples.We recommend having separate pipettes and pipette tips for PCR setup and post-PCR analysis. Use sterile, filter tips for pipetting to minimize contamination from aerosols. Use 10% bleach to clean micropipettes regularly.Addition of the template and subsequent handling of the amplified product should be away from the PCR set up hood, in an area easy to decontaminate. Decontaminate PCR work areas regularly with 10% bleach and UV irradiation. Use separate dedicated PCR work areas for reaction setup preferably in a PCR set up hood in a clean room that are well away from any possible nucleic acid contamination.Always include appropriate positive and negative controls in your experiment. This will decrease the chances of contamination, allow you to start with a fresh tube if you do have contamination, and minimize freeze–thaw cycles that can reduce oligonucleotide quality. Aliquot your probe and primers into the volumes required for a single experiment.Use water, tubes, and reagents that you are sure to be sterilized (see IDT buffers and solutions).General suggestions for avoiding false positives in your negative template control (NTC) sample Target Capture Probe Design & Ordering Tool.Library Concentration Conversion Calculator.Alt-R Predesigned Cas9 crRNA Selection Tool.SYBR Green dye assay and PrimeTime probe assays.PCR Allele Competitive Extension (PACE) genotyping.Shotgun metagenomics for infectious diseases.Drug target identification via CRISPR screening. ![]()
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