Need to verify that the DNased RNA I made previously does not have any detectable gDNA present.
Ran the withering syndrome qPCR assay on the DNased RNA.
Standard curve was p18RK7 from 20161128.
All samples were run in duplicate. As such, the number of samples required to qPCR runs.
Master mix calcs are here (Google Sheet): 20170406_qPCR_WSN1_capstone
Plate layout, cycling params, etc. can be found in the qPCR Report (see Results).
Baseline threshold was manually set to 580, as previously determined by Lisa.
qPCR Report (PDF): Sam_2017-04-06 11-36-53_CC009827_capstone_RLO_viability_WSN1.pdf
qPCR Data File (CFX96): Sam_2017-04-06 11-36-53_CC009827_capstone_RLO_viability_WSN1.pcrd
Well, some samples came up positive for residual DNA. The samples that came up positive are all three dilutions of the RLO used for initial infection of the abalone.
This makes things interesting to deal with. Seeing that no other samples have detectable DNA suggests that those samples are fine to move forward with for reverse transcription. However, it’s unlikely that the DNase treatment only worked on a subset of a samples, since it was distributed via a master mix.
Regardless, there isn’t any additional RNA to work with. So, I’ll put the samples that came up positive through a second round of DNase treatment. Addtionally, I may dilute them slightly to avoid complications from accumulation of too much DNase buffer, due to leftover buffer from the first round…
Amplification Plots from Sam_2017-04-06 10-01-23_CC009827.pcrd
Green = p18RK7 standards
Blue = samples
Red = No template control
Standard Curve from Sam_2017-04-06 10-01-23_CC009827.pcrd
Amplification Plots from Sam_2017-04-06 11-36-53_CC009827_capstone_RLO_viability_WSN1.pcrd
Standard Curve from Sam_2017-04-06 11-36-53_CC009827_capstone_RLO_viability_WSN1.pcrd