I am trying to optimize the 1-step RT-ddPCR system using Invitrogen™ SuperScript™ III One-Step RT-PCR master mix Kit for absolute virus quantification of the virus. But in most of the articles, they use a Bio-rad one-step rt-ddPCR supermix probe kit.
however, in one article superScript™ III One-Step RT-PCR master mix Kit was used for 1 step RT- ddPCR assay and they succeeded.
I have done a couple of experiments using the Invitrogen kit and I got ABS quantification in a few experiments. But I am facing trouble setting up the detection limit and I don't know whether it is because of the supermix kit or my skill!
I am seeking some suggestions from experts. Thank you.
hi why u mixing the steps .you must making share from master mix .
If you're brave enough to risk putting a $40K to $300K droplet reader out of service, you may be able to set up your reactions using SuperScript™ III One-Step RT-PCR master mix, and defined it as whatever master mix you want in the software. I'd give it pretty good chances of working.
You imply it has been working in Absolute Quantification mod (ABS), but you're having issues with the detection limit. Is your lowest tested level of loading under 50 copies/well, and what seems to be the issue? Is the detection inaccurate or imprecise? Near the LOD, ddPCR data frequently becomes hyper variable.
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