Performed qPCR on the DNased oyster RNA from earlier today with Gigas_18s_F/R primers to verify removal of detectable gDNA in the samples, since the qPCR from 20090508 indicated residual gDNA was still present in some of the DNase treated RNA. Plate layout/set up is here.
Results: Samples BB# 2, 5, 6, 15 still came up as positive for gDNA contamination. These will NOT be used to make cDNA for subsequent qPCRs.
Followed the rigorous protocol for Ambion’s Turbo DNA-free protocol for the following RNAs:
BB#11-20
DH#11-20
Used 10ug of RNA (200ng/uL) in 50uL as directed. Here are the calcs for FF and DH.
Followed standard protocol on DNased samples from 20090508:
BB#1-10
DH#1-10
The standard protocol should be fine for these samples, since the procedure worked on Friday for some of them.
Isolated gDNA according to Molecular Research Center TriReagent protocol from BB#1-20 and DH#1-20. Resuspended DNA in 600uL of 8mM NaOH. Spec.
Results: HORRIBLE! This is some of the worst “DNA” I’ve ever seen. Peaks everywhere EXCEPT at 260nm. Here’s a link to the actual numbers. It’s a text file and is comma separated, so you should open with Excel for it to be readable.
Spoke with Steven. Will pursue RNA instead of continuing down this path for now.
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.
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.
All of the RNA (50uL) was DNase treated with Ambion’s Turbo DNA-free kit according to protocol. Samples were spec’d.
Results:
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.
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.
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.
