This question is for Taqman qPCR assay containing FAM/TAMRA probe for amplification of Viral RNA.
While pipetting RNA/cDNA in 96-well plate, I always mix it couple of times to ensure mixing of qPCR reagents and RNA/cDNA. I have observed it to be a major factor for contamination. Has anyone tried not mixing, just pipetting RNA/cDNA, sealing the plate and then vortexing (slow speed) to achieve the same purpose?
We do the same for digital droplet PCR, I cant see why this cant be replicated with Taqman qPCR as well!
Thank you, any comments/feedback much appreciated!
Hello, Brijen!
I do it! Pipette the samples, seal the plate, vortex at low speed, and then spin to lower the droplets to the bottom of the plate. The amplification curves look good!
I could be in the minority, because I don't mix the cDNA with qPCR reagents multiple times. As you mentioned, I too realized that mixing the prep a few times can introduce contamination and also allow for some of the reaction mix to stick to the pipette tip. So I usually add the reagents in one go and gently centrifuge the plates to collect the droplets. I haven't had any issues with results thus far. I assume that the mixture gets agitated as it gets close to boil during each melting phase of the thermocycler.
But I do gently pipette the stock master mix containing all the reagents (minus the template) before aliquoting into the qPCR tubes. This is just to make sure that the enzyme is homogenous and not pooled at the bottom.
Praveesh Valissery Thank you for the feedback. I will try it out. It makes sense
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