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Yes. We have recently confirmed that primers of lengths typical for use in PCR (e.g. 18-23 nucleotides) work just fine in RPA in many cases. Note though that kinetics can be a little slower, so if you wish to take full advantage of the potential of RPA for rapid amplification within 10-15 minutes we recommend using slightly longer primers (as …
In practise, it makes no difference which way round the labels are used i.e. which label is on the probe, and which one is on the primer. However, for ease of further test development, it may prove useful to put the hapten for the conjugate antibody on the probe (FAM for Milenia, Biotin for PCRD). This way, users who proceed …
Ideally the conditions of the screen should mimic as closely as possible the conditions expected in the final assay (approximate template copy number, sample purity, depth of multiplexing, etc.).
For further details on primer design, please see the assay design manual
If you are combining your RNA template with MgOAc before adding it to your reactions, you may be stabilising RNA tertiary structures that inhibit the reverse transcriptase. Adding the RNA to your reaction mixture either before you add the MgOAc, or spatially separating it (i.e. adding RNA and MgOAc to opposite sides of your reaction vessel or lid) and adding …
This is normal for TwistAmp® Liquid reactions, and is not a problem. RPA reactions form emulsions which can sometimes be observed by the naked eye.
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As RPA reactions are performed at a constant temperature and under conditions under which the melting behaviour of DNA is drastically altered by DNA melting proteins, conventionally calculated melting points are not directly applicable to the system.
The precise rules for this are not yet known, this is why primer screening is so important.