Tag Archives: Turbo DNA-free

DNase Treatment – Ronit’s C.gigas Ploiyd/Dessication Ctenidia RNA

After quantifying Ronit’s RNA earlier today, I DNased them using the Turbo DNA-free Kit (Ambion), according to the manufacturer’s standard protocol.

Used 1000ng of RNA in a 50uL reaction in a 0.5mL thin-walled snap cap tube. Samples were mixed by finger flicking and then incubated 30mins @ 37oC in a PTC-200 thermal cylcer (MJ Research), without a heated lid.

DNase inactivation was performed (0.1 volumes of inactivation reagent; 5uL), pelleted, and supe transferred to new 1.7mL snap cap tube.

Samples were stored on ice in preparation for qPCR to test for residual gDNA.

DNase calculations are here:

Samples will be permanently stored here (Google Sheet):

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.

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.