If positive results are seen in no template controls it is commonly caused by contamination, particularly, if the positive control oligonucleotides provided in the kit display false positives when no template is added. Contamination can occur if you are pipetting high copies (10^6 /µl or above) of template in the same area as carrying out RPA testing. We strongly recommend having separate pre and post amplification areas i.e. where you set up reactions, and where you open them for analysis. Other contamination control measures that we recommend include aliquoting primers and probes with different pipettes, and in a different lab, to where you perform RPA experiments. When conducting initial RPA assay testing, we suggest working with low amounts of template (at ~50 copies/µl) and advise against opening anything with more than 10,000 copies/µl anywhere near where you do set-ups if possible. Another good contamination control measure we would suggest adopting is to add the MgOAc to the lids, and spinning it in to start your reactions. It is essential to apply strict contamination control measures when using any end point detection methods where tubes of reactions are opened post amplification. To rule out, or clear contamination we suggest decontaminating work areas with 10% bleach, and using fresh reagent aliquots (buffer, MgOAc, etc). You may need to repeat this bleach clean-up a few times before removing contamination completely.
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