Ran qPCR with the p16RK7 curve using the new ABI reagents to test SPUD performance now that we have a fully functional withering syndrome qPCR assay again.
Also repeated the following 2010 Water Filter DNAs that were run yesterday, due to lack of amplification in one of the duplicates (likely due to very late amplification):
- SNI Site 8 +100M #1
- SNI Site 3 -100M #1
- SNI Site 3 -500M #1
Master mix calcs are here: 20140423 – qPCR p16RK7 Curve WSN1 SPUD
Plate layout, cycling params, etc. can be found in the qPCR Report in the Results below.
For Lisa’s samples, only the SNI Site 3 -100M #1 showed any amplification (mean Cq of 39.31).
The SPUD assay still shows inhibition at higher concentrations of the standard curve (>= 3e4 copies). Possible that the SPUD probe is dying (the working stock has been through many freeze/thaw cycles which could definitely be affecting its performance…). This is slightly disconcerting, but I’m just happy that the standard curve is functional once again!!