Used EtOH precipitated BB01 RNA from 20110216 and followed Ambion’s “rigorous” protocol, utilizing a total of 2uL of DNAse. Briefly, samples were incubated @ 37C for 30mins, an additional 1uL of DNase was added to each sample, mixed and incubated for an additional 30mins @ 37C. After finishing protocol, samples were spec’d.
First reading had an air bubble and should be ignored. DNased RNA looks good, based on 260/280 ratios. As is usually the case for DNased RNA, the 260/230 ratios are on the low side. Will check DNased RNA for residual gDNA.
Due to residual gDNA contamination, will EtOH precipitate in order to treat with DNase again. Add 0.5 vols 3M NaAOc (pH = 5.2), 2.5 vols 100% EtOH, mixed and incubated @ -20C for 30mins. Pelleted RNA @ 16,000g, 4C 30mins. Washed RNA with 1mL 70% EtOH (2x due to fear of residual salts from DNase Buffer). Pelleted RNA @ 16,000g, 4C, 15mins. Resuspended RNA in 45uL nuclease-free H2O. Sample was stored @ -80C (in “Sam’s RNA Box #1) until it could be DNased again.
Ran qPCR on DNased RNA from earlier today to verify that it was free of contaminating gDNA. Used C.gigas 18s primers (SR IDs: 156, 157). 0.5uL (50ng) 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”.
Need more DNased RNA to finish repeating of PROPS. Some samples had insufficient quantities of DNased RNA remaining in BB01 and BB09. Used 10ug of each RNA and followed Ambion’s “rigorous” protocol, utilizing a total of 2uL of DNAse for each sample. Briefly, samples were incubated @ 37C for 30mins, an additional 1uL of DNase was added to each sample, mixed and incubated for an additional 30mins @ 37C. After finishing protocol, samples were spec’d.