Tag Archives: DNase

DNase Treatment – O.lurida Ctenidia 1hr Post-Mechanical Stress RNA

Quantified the RNA I isolated from Jake’s samples on 20150715 and 20150710 using the Roberts Lab NanoDrop1000 (ThermoFisher).

 

 

 

Overall, the yields are good. The 260/280 ratios are mediocre. Will proceed with DNase treatment.

DNased 1.5ug of RNA from each sample using the Turbo DNA-free Kit (Ambion/Life Technologies), following the “rigorous” protocol.

Briefly:

  • 50μL reactions were carried out in 0.5mL tubes
  • added 1μL of DNase to each tube
  • incubated 30mins @ 37C
  • added additional 1μL of DNased
  • incubated 30mins @ 37C
  • added 0.2 vols (10.2μL) of DNase Inactivation Reagent
  • incubated and mixed for 2mins @ RT
  • spun 1.5mins, 10,000g @ RT
  • transferred 50μL of supe to sterile 1.5mL snap cap tubes
  • spec’d on Roberts Lab NanoDrop1000

Samples were stored @ -80C in Shellfish RNA Box #6. Will quantify at a later date.

DNase reaction calcs: 20150727_Jake_Oly_mech_stress_DNase_calcs

 

DNase Treatment – Jake’s O.lurida Ctenidia RNA (1hr Heat Shock) from 20150506

Since the O.lurida RNA I isolated on 20150506 showed residual gDNA via qPCR, I treated 1.5μg of RNA from each sample using the Turbo DNA-free Kit (Ambion/Life Technologies), following the “rigorous” protocol.

Briefly:

  • 50μL reactions were carried out in 0.5mL tubes
  • added 1μL of DNase to each tube
  • incubated 30mins @ 37C
  • added additional 1μL of DNased
  • incubated 30mins @ 37C
  • added 0.2 vols (10.2μL) of DNase Inactivation Reagent
  • incubated and mixed for 2mins @ RT
  • transferred 50μL of supe to sterile 1.5mL snap cap tubes
  • spec’d on Roberts Lab NanoDrop1000

Samples were stored @ -80C in Shellfish RNA Box #5 and Box #6.

DNase reaction calcs: 20150514_Jake_Oly_1hr_HS_DNase_calcs

 

 

Results:

Google Spreadsheet: 20150514_DNased_RNA_Jake_Oly_1hr_HS_ODs

 

 

 

 

All samples look pretty good except for HT1 8 (RNA concentration is ridiculously high!) and NT1 8 (RNA concentration is way below expected). Will check for residual gDNA via qPCR.

DNase Treatment – Jake’s O.lurida Ctenidia RNA (Controls) from 20150507

Since the O.lurida RNA I isolated on 20150507 showed residual gDNA via qPCR, I treated 5μg of RNA from each sample using the Turbo DNA-free Kit (Ambion/Life Technologies), following the “rigorous” protocol.

Briefly:

  • 50μL reactions were carried out in 0.5mL tubes
  • added 1μL of DNase to each tube
  • incubated 30mins @ 37C
  • added additional 1μL of DNased
  • incubated 30mins @ 37C
  • added 0.2 vols (10.2μL) of DNase Inactivation Reagent
  • incubated and mixed for 2mins @ RT
  • transferred 50μL of supe to sterile 1.5mL snap cap tubes
  • spec’d on Roberts Lab NanoDrop1000

Samples were stored @ -80C in Shellfish RNA Box #5 and Box #6.

DNase reaction calcs: 20150514_Jake_Oly_control_DNase_calcs

 

 

Results:

 

Google Spreadsheet: 20150514_DNased_RNA_Jake_Oly_controls_ODs

 

 

 

 

Overall, samples look fine. Will check for residual gDNA via qPCR.

qPCR – DNased Manila Clam Larvae RNA (from August 2012 – Dave’s Notebook)

Performed qPCR on Dave’s manila clam larvae DNased RNA from August 2012 using EF1a primers (SR IDs: 1463, 1474).

Master mix calcs are here. https://docs.google.com/spreadsheet/ccc?key=0AmS_90rPaQMzdHc5amwzZzdDa1d0VXQzLVU0WkFTc0E

Plate layout, cycling params, etc can be found in the qPCR Report (see Results).

Positive control was pooled cDNA taken from Dave’s cDNA plate on 8/7/2012.

Results:

qPCR Data File(CFX96) http://eagle.fish.washington.edu/Arabidopsis/qPCR/CFX96/Roberts%20Lab_2012-10-26%2010-48-07_CC009827.pcrd

qPCR Report(PDF) http://eagle.fish.washington.edu/Arabidopsis/qPCR/CFX96/Roberts%20Lab_2012-10-26%2010-48-07_CC009827.pdf

Here’s a quick Google Spreadsheet summary highlighting samples that came up positive/negative.

https://docs.google.com/spreadsheet/ccc?key=0AmS_90rPaQMzdFFHb3YwWE01UG00TnY3OWo2cWx2UVE

Approximately half of the samples (~27) came up positive for still having gDNA in them.

There are three pCO2 treatments: 1000ppm, 750ppm, and 400ppm. There are six sampling dates: 7/29/2011, 8/2/2011, 8/9/2011, 8/12/2011. Currently, it is unknown when the Day 0 samples were collected. Have emailed Dave for deets.

There are only two dates (7/29/2011 and 8/5/2011) that have a full set of samples (i.e. 1000ppm, 750ppm and 400ppm) that exhibit DNA-free RNA. Will discuss with Steven on how to proceed.

UPDATE 20121031 – Dave emailed and indicated the experimented started on 7/27/2011. Additionally, the two sample sets that are complete are Day 2 and Day 7. Discussing with Steven, we have decided to run a few genes and see how the expression levels compare to the NGS data analysis for these samples. If the qPCR data supports the NGS data, then that information will be relayed to the BMC Genomics reviewers in response to their critiques. A copy of the manuscript is here(may not be publicly viewable). https://docs.google.com/document/d/1Ii1lODz2oThiyxZtHBblUEdzyhIVq92n8jkEjhkuuts/edit

DNase Treatment – C.gigas Larvae RNA from yesterday

Treated 5ug of total RNA (in a 50uL rxn) using Turbo DNA-free (Ambion) according to the “Standard” protocol. Samples were spec’d on the Roberts Lab NanoDrop 1000.

Results:

All samples look good, both quality and quantity-wise. Will check for residual gDNA in these samples via qPCR.

RNA Isolation – Dave’s Manila Clam (Venerupis philippinarum) Gill Samples (#25-48)

Isolated RNA from Manila Clam gill samples provided by Dave, according to protocol. Samples were resuspended in 0.1%-DEPC H2O and spec’d on the Roberts Lab NanoDrop1000. Samples were stored @ -80C in Dave’s box that the tissue was initially stored in.

Results:

All samples look great with excellent yields and great 260/280 values. Will proceed with DNasing. (Note: Sample #42 appears twice because the first reading had an air bubble and, as such, should be discarded.)

 

DNased RNA using Ambion’s Turbo DNA-free Kit following the “routine” protocol. 5ug of total RNA from each sample was treated in 50uL reactions. Samples will be spec’d on Monday with the Roberts Lab NanoDrop 1000.

Results:

DNAse – C.gigas RNA from 20120124

5ug of each RNA was DNased using Ambion’s Turbo DNA-free Kit, according to the rigorous protocol and spec’d on Roberts Lab NanoDrop 1000. RNA volume calcs are here.

Results:

DNased RNA looks fine. Low OD260/280 ratios, but this is often seen after DNase treatment and particularly with low [RNA]. Will perform qPCR to assess gDNA removal.

DNase – Hard Clam Gill RNA (from earlier today)

DNased 5ug of RNA from each sample with Ambion’s Turbo DNA-free Kit, according to the manufacturer’s rigorous protocol. Samples were spec’d and stored @ -80C in the “Hard Clam V.t. Experiment RNA” box.

Results:

Due to lack of a positive control (and lack of primers known to amplify gDNA), these DNased RNA samples will NOT be checked to verify elimination of gDNA carryover at this time. Will proceed with making cDNA for qPCR anyway.

DNase – C.gigas BB01 (PROPS) RNA (from 20090507)

Since the previous DNase treatment failed for this sample, will repeat but will start with less RNA (5ug instead of 10ug). Need more DNased RNA to finish repeating of PROPS. Some samples had insufficient quantities of DNased RNA remaining in BB01. Used 5ug of 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): 5ug/1.824ug/uL = 2.74uL RNA + 42.26uL H2O (to 45uL) + 5uL 10X DNase Buffer = 50uL

Results:

RNA looks OK, based on OD260/280. Would like that value to be higher, though. OD260/230 is low, which is typical post-DNased treatment. Will check for residual gDNA via qPCR.