Pcr Contaminants

When performing PCR amplification, it is always important to be aware of potential sources of DNA contamination. The power to amplify very small amounts of DNA to detectable levels demands that special care be taken to prevent cross-contamination between different samples. This is especially true for PCR targets expected to be present in low numbers because greater efforts are usually required to amplify these sequences. Sources of contamination include (1) genomic DNA contaminating RNA samples, (2) cross-contamination among different nucleic acid samples processed simultaneously, (3) laboratory contamination of cloned target sequences (genomic or cDNA), and (4) carryover of PCR products. In general, the likelihood of contamination is substantially reduced by working in a clean laboratory and using good laboratory practices (wearing clean gloves at all times). Carryover products from other PCR reactions can be effectively controlled by the use of aerosol-free pipet tips, by using dedicated pipettors and solutions, and by maintaining separate areas to handle pre-PCR and post-PCR solutions and samples. In addition to these simple precautions, certain PCR strategies can reduce the possibility of drawing conclusions from false-positive results. For instance, in RT-PCR applications, the use of oligonucleotide primers that are positioned within different exons of the gene of interest will facilitate distinction of PCR products from the RNA template vs amplicons resulting from the amplification of contaminating genomic DNA. In this example, PCR products derived from genomic DNA will be of a different size (larger) than those from cDNA. In all PCR applications, it is essential to include proper positive and negative control reactions to guard against systematic contamination of PCR reagents and to ensure that the desired amplicon is produced in positive reactions.

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