Used EtOH precipitated BB01 RNA from 20110225 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.
Results:
RNA looks good, based on the OD260/280. As usual after DNasing, the OD260/230 is on the low side. Will check for residual gDNA via qPCR.
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.
Results:
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.
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.
DNase Rxn Calcs:
BB01 (1.824ug/uL): 10ug/1.824ug/uL = 5.48uL RNA + 39.52uL H2O (to 45uL) + 5uL 10X DNase Buffer = 50uL
BB09 (0.506ug/uL): 10ug/0.506ug/uL = 19.77uL RNA + 25.23uL H2O (to 45uL) + 5uL 10X DNase Buffer = 50uL
Results:
260/280 values look great. 260/230 values look bad, but this is not unusual for samples post-DNase treatment.
Pooled 2ug of each sample in each group (MAX, CA, MA) for a total of 6ug of RNA (3 total samples), brought volume up to 50uL and DNased using Ambion’s Turbo DNA-free following the rigorous protocol. Calcs can be seen here. Spec’d:
Results:
All samples look pretty good. Oddly, the 260/280 ratios are absolutely perfect, despite the 260/280 ratios from each individual sample being less than stellar (see yesterday’s EtOH precipiation). Also of note is that the concentrations for all three samples are extremely close, reflecting the accuracy of the NanoDrop readings of each individual sample used for the pool as well as my pipetting. 
RNA was stored @ -80C in “Sam’s RNA Box #1.”
Recovered ~50uL from each sample which means each pool yielded ~3.85ug of RNA after DNase treatment. Will proceed with making cDNA from these three pools. In the interest of time (and the failure of our Opticon), I will not verify that these do NOT still contain gDNA (and, it’s pretty unlikely that they do).
DNase Treatment – Sepia RNA (from 20091204)
Samples were DNase treated with Ambion’s Turbo DNA-free kit, following the rigorous protocol. Used 6uL of each sample, brought up to 50uL with H2O, added 5uL of 10x buffer and 1.5uL of DNase. Incubated 37C for 30mins, added an additional 1uL of DNase and incubated @ 37C for 30mins. Added 0.2 volumes of DNase Inactivation reagent and incubated at RT for 2mins with regular mixing. Spec’d RNA.
Results:
Set up reverse transcription rxns using 200ng of each DNased RNA, using Promega oligo dT primers and M-MLV Reverse Transcriptase according to Promega protocol. RNA/Oligo dT primer workup here. Primer and DNAsed RNA were mixed and brought up to 18.25uL with H2O. Samples were heated @ 70C for 5mins and then placed immediately on ice. The RT master mix set up can be found here. 6.75uL of the RT master mix was added to each tube, mixed, spot spun and then incubated @ 42C for 1hr, heat inactivated @ 95C for 3mins and stored @ 4C.
5 samples from yesterday’s treatment still came up on qPCR, so I will re-DNase those 5 samples. Followed standard protocol in Ambion’s Turbo DNA-free kit.
Used 5uL of RNA from each sample, brought samples up to 50uL with H2O and treated according to Ambion’s Turbo DNA-free kit. Rigorous protocol was followed (1.5uL DNase initially + 1.5uL additional DNase after 30mins). Transferred treated samples to a PCR plate to facilitate further manipulation of the samples. Will perform qPCR on these samples to make sure treatment worked.
Used 5uL of RNA from each sample, brought samples up to 50uL with H2O and treated according to Ambion’s Turbo DNA-free kit. Transferred treated samples to a PCR plate to facilitate further manipulation of the samples. Will perform qPCR on these samples to make sure treatment worked.
DNase treated Abalone Dg RNA. Followed Ambion’s Turbo DNA-free Kit rigorous protocol. Here are the calcs/workup for the DNase treatment. This used previously untreated RNA.
DNase treated the 07:12 set of Abalone Dg RNA. Also, respec’d the RNA prior to proceeding. Followed Ambion’s Turbo DNA-free Kit rigorous protocol. Here are the calcs/workup for the DNase treatment. This used previously untreated RNA.
