Previuosly checked existing crab RNA for residual gDNA on 20200226 and identified samples with yields that were likely too low, as well as samples with residual gDNA. For those samples, was faster/easier to just isolate more RNA and perform the in-column DNase treatment in the ZymoResearch Quick DNA/RNA Microprep Plus Kit; this keeps samples concentrated. So, I isolated more RNA on 20200306 and now need to check for residual gDNA.
Used 2ng of RNA (1uL) in each reaction. A 5uL dilution of each sample was made to a concentration of 2ng/uL. The decision for this quantity was based on the amount of RNA we might use in a reverse transcription reaction (50ng/25uL) and the volume of the resulting cDNA we’d run in a qPCR reaction (1uL). Calculations and the qPCR results (Cq values) are below (Google Sheet).
All reactions were run with 2x SsoFast EVA Green qPCR Master Mix (BioRad) on the Roberts Lab CFX Connect qPCR machine.
All samples were run in duplicate. See the qPCR Report (in Results section below) for plate layout, cycling params, etc.
Master mix calcs are here (Google Sheet):
qPCR data file (CFX):
qPCR Report (PDF):
qPCR results file (CSV):
Positive control amplified.
No template controls (NTCs) did not amplify.
No amplification in any samples.
Things look good. Will evaluate which samples will be used for reverse transcription and subsequent qPCRs.
Blue: RNA samples
Green: Positive control gDNA
Red: No template controls
Melt curve plots
Same color scheme as amplification plots