Yesterday’s qPCR indicated that all of the RNA still contained gDNA contamination. So, took 10ug of RNA from BB #1-10 and DH#1-10 (calcs/workup BB and DH) and brought the volumes up to 50uL with 0.1% DEPC-H2O. Processed the samples according to Ambion Tubrbo DNA-free protocol. Will proceed with qPCR immediately.
Category Archives: PROPS
qPCR – DNased oyster RNA from earlier today
Performed qPCR on the DNased RNA to with Gigas_18s_F/R primers to verify removal of detectable gDNA in the samples. Plate layout/set up can be found here.
Results: All samples produced a signal. In retrospect, this is likely due to having too much RNA for the DNase treatment. I proceeded with the DNase treatment and qPCR prior to specing the samples. Spec revealed that most of them were highly concentrated; more than the Ambion protocol recommends. Will redo the DNase treatment on a subset of the samples using the appropriate quantity of RNA.
DNase Treatment – Oyster RNA from today
All of the RNA (50uL) was DNase treated with Ambion’s Turbo DNA-free kit according to protocol. Samples were spec’d.
Results:
RNA Isolation – Mac’s oyster tissues (BB and DH) (CONTINUED from yesterday)
Samples were precipitated according to TriReaget protocol. RNA was resuspended in 50uL of 0.1% DEPC-H2O and then heated @ 55C for 10mins to help dissolve the pellets. Samples will be DNase treated.
RNA Isolation – Mac’s oyster tissues (BB and DH) (CONTINUED from yesterday)
Completed the reaminder of the samples (BB#9-20 and DH#1-20) up to the point of precipitation. Isopropanol was added and stored @ -20C. Organic phase was retained for subsequenct gDNA isolation and stored @ 4C.
RNA Isolation – Mac’s oyster tissues (BB and DH)
Processed BB#1-8 up to the point of precipitation. Added isopropanol and stored @ -20C. Organic phase was retained for subsequent gDNA isolation and stored @ 4C.