Isolated RNA from Jake’s Olympia oyster ctenidia, controls, collected on 20150422. Samples had been homogenized and stored @ -80C.
The following sample tubes (heat-shocked oyster ctenidia) were removed from -80C and thawed at RT:
42215 HC 1
42215 HC 2
42215 HC 3
42215 HC 4
42215 HC 5
42215 HC 6
42215 HC 7
42215 HC 8
42215 NC 1
42215 NC 2
42215 NC 3
42215 NC 4
42215 NC 5
42215 NC 6
42215 NC 7
42215 NC 8
42215 SC 1
42215 SC 2
42215 SC 3
42215 SC 4
42215 SC 5
42215 SC 6
42215 SC 7
42215 SC 8
NOTE: 0.1% DEPC-H2O used throughout this procedure was prepared on 7/15/2010 by me.
According to Jake’s notebook entry, the samples should have been previously homogenized in RNAzol RT (Molecular Research Center; MRC). However, none of the samples showed evidence of being homogenized:
(http://eagle.fish.washington.edu/Arabidopsis/Pics/20150507_Jake_Oly_tissue_RNAzol.JPG)
Procedure:
Samples were homogenized with disposable pestle in their respective tubes and vortexed.
Added 400μL of 0.1% DEPC-H2O to each sample and vortexed 15s.
Incubated samples 15mins at RT.
Centrifuged tubes 15mins at RT @ 16,000g.
750μL of the supe was transferred to a clean tube, added equal volume of isopropanol (750μL), mixed by inversion (20 times), and incubated at RT for 15mins.
Centrifuged 12,000g for 10mins.
Discarded supe.
Washed pellets with 500μL of 75% EtOH (made with 0.1% DEPC-H2O) and centrifuged 4,000g for 3mins at RT. Repeated one time.
Removed EtOH and resuspended in 100μL of 0.1% DEPC-H2O. Most samples required vortexing to dissolve pellet.
Sample tubes were transferred to ice, quantified on the Roberts Lab NanoDrop1000, and stored @ -80C in their original box, pictured:
(http://eagle.fish.washington.edu/Arabidopsis/Pics/20150507_Jake_Oly_RNA_box.JPG)
Results:
(http://eagle.fish.washington.edu/Arabidopsis/20150507_Jake_oly_control_RNA_ODs.JPG)
(http://eagle.fish.washington.edu/Arabidopsis/20150507_Jake_oly_control_RNA_plots-01.JPG)
(http://eagle.fish.washington.edu/Arabidopsis/20150507_Jake_oly_control_RNA_plots-02.JPG)
Google Spreadsheet with absorbance data: 20150507_Jake_Oly_control_RNA_ODs
Excellent yields and pretty solid 260/280 ratios (>1.85). Interestingly, the 260/230 ratios aren’t so great (compared to yesterday’s isolations). I suspect that the reason for this is that there appeared to be more starting tissue in these samples than yesterday’s. The greater quantity of tissue explains the higher yields and could be tied to the decrease in the 260/230 ratios…
Anyway, things look good. Next step will be to check for gDNA carryover in these samples and yesterday’s samples.