After DNasing Ronit’s RNA earlier today, I needed to check for any residual gDNA.
Identified some old, old C.gigas 18s primers that should amplify gDNA:
- gigas18s_fw (SRID 157)
- gigas18s_rv (SRID 156)
Used some old C.gigas gDNA (BB15 from 20090519) as a positive control.
Samples were run on Roberts Lab CFX Connect (BioRad). All samples were run in duplicate. See qPCR Report (Results section) for plate layout, cycling params, etc.
qPCR master mix calcs (Google Sheet):
qPCR Report (PDF):
qPCR File (PCRD):
qPCR Data (CSV):
Well, this primer set and/or the gDNA is not good. In the plots below, the positive control gNDA is in green, samples in blue, and no template controls (NTC) are in red.
Poor performance is most easily noticed when looking at the melt curves. They have multiple peaks, suggesting non-specific amplification, even in the positive control.
Additionally, although less evident from just looking at the plots, is the replicates are highly inconsistent. Although it’s possible that might be due to poor technique, it’s very unlikely.
Will have to identify different primers and/or positive control DNA.