Ran qPCR on DNased RNA from earlier today to verify removal of contaminating gDNA. Used C.gigas 18s primers (SR IDs: 156, 157). 0.5uL (~40ng) of DNased RNA was used for testing. This corresponds, roughly, to the amount of sample that would be carried through to qPCR analysis of cDNA, assuming 1ug of RNA was used to make the cDNA (cDNA = 1000ng RNA/25uL = 40ng/uL, 1uL of cDNA in 25uL qPCR reaction). Positive control sample was ~25ng BB16 gDNA (from 20090519). Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results). RNA was stored @ -80C in “Sam’s -80C Box”.
[ qPCR Report (PDF)(https://eagle.fish.washington.edu/Arabidopsis/qPCR/Roberts%20Lab_2011-03-01%2013-10-22_CC009827.pdf)
[ qPCR Data File (CFX96)(https://eagle.fish.washington.edu/Arabidopsis/qPCR/Roberts%20Lab_2011-03-01%2013-10-22_CC009827.pcrd)
Residual gDNA is present in the sample. So, it’s become apparent that it’s virtually impossible to rid the BB01 RNA of contaminating gDNA. Will discuss with Steven and Mac if it’s feasible to exclude this from the additional PROPS analysis that needs to be done and how this could potentially affect our data. Talked to Steven and, duh, we can just remove the previous BB01 data from our analysis. Will make new batch of cDNA from existing DNased RNA samples.