See Rachel’s 441 Notebook from 10/28/2009 through 11/4/2009 for experiment info. 500mL of water was collected from the CO2 and the air tanks. Water was vacuum filtered through Watman paper. The Watman paper was allowed to dry over the weekend.
Results:
CO2 = 35.9mg
Air = 14.4mg
Submitted 400ng of liver mRNA (SR01) and 400ng of testes (gonad) mRNA (SR02) to MoGene. Samples were packed in dry ice and FedEx.
UPDATE 20091028 FedEx had a shipping delay and the samples did not get delivered today. Will be delivered tomorrow. We will be credited the full shipping amount for this delay.
Sample was spun 16,000g, 30mins, 4C. Supe removed. Pellet washed with 1mL 70% EtOH. Spun 16,000g, 10mins, 4C. Supe removed. Pellet resuspended in 250uL of nuclease free H2O. Will proceed with mRNA isolation.
Isolated mRNA using Ambion’s MicroPolyA Purist Kit according to protocol. Performed two rounds of isolation to decrease residual rRNA carryover that we frequently see after a single round.
Results:
Started with ~90ug of total RNA. Yield of mRNA = 3.26ug. That is a ~3.6% recovery of mRNA.
A subset (3 samples from each group) of samples were pooled (see spreadsheet, green-highlighted samples), each providing ~7.5ug of RNA, yielding 112.17uL. 0.1 vols of 3M NaOAC, pH = 5.2 were added to the tube (11.22uL). 2 vols of EtOH (246.8uL) was added to the tube. Tube was vortexed to mix and incubated @ -20C O/N.
Isolated mRNA using Ambion’s MicroPolyA Purist Kit according to protocol. Performed two rounds of isolation to decrease residual rRNA carryover that we frequently see after a single round.
Results:
Started with ~500ug. Total yield = 5.3ug. That is a 1.06% recovery of mRNA.
A subset of samples (4 samples from each group) were pooled (see spreadsheet, green-highlighted samples), each providing ~68ug of RNA. The pooled sample was split into two tubes (435uL/tube). 0.1 vols (43.5uL) of 3M NaOAC (pH=5.2) were added, then 2 vols of 100% EtOH (957uL). Tubes were vortexed and incubated @ -80C for 30mins. RNA was pelleted by spinning 16,000g, 30mins, 4C. Supe was removed, pellets washed with 70% EtOH. Tubes were spun 16,000, 10mins, 4C. Supe removed. Pellets were resuspended in 250uL of RNase-free H2O and stored @-80C until Monday for mRNA isolation.
RNA was isolated according to protocol. Pellets were resuspended in 50uL of 0.1%DEPC-H2O, heated @ 55C for 5 mins, spec’d and stored @ -80C in the “Herring RNA Box #1″.
Results:
Most of the samples look good, however there are a number of samples that are downright bad. Either no RNA or very low concentrations with poor 260/280, 260/230 ratios.
From the Seeb Lab. Homogenized entire gonad/ovary samples in 5mL of TriReagent with the sonicator. In essence, based on the manufacturer’s recommendation, this means the ratio of tissue:TriReagent was ~2x. Transferred 0.5mL of homogenized gonad/ovary sample to 1.5mL snap cap tubes and added an additional 0.5mL of TriReagent, to adjust the ratio of tissue:TriReagent to ~1x. Samples were then stored @ -80C. These will be further processed tomorrow.
RNA was isolated according to protocol. Pellets were resuspended in 200uL of 0.1%DEPC-H2O, heated @ 55C for 5 mins, spec’d and stored @ -80C in the “Herring RNA Box #1″.
Results:
RNA looks good. Will speak with Steven how to proceed and whether or not to check for gDNA carryover.
From Seeb Lab. Homogenized entire liver samples in 5mL of TriReagent with the Tissue Tearers. In essence, based on the manufacturer’s recommendation, this means the ratio of tissue:TriReagent was ~2x. Transferred 0.5mL of homogenized liver sample to 1.5mL snap cap tubes and added an additional 0.5mL of TriReagent, to adjust the ratio of tissue: TriReagent to ~1x. Samples were then stored @ -80C. These will be further processed once all remaining liver samples have been homogenized inTriReagent.