Tag Archives: NanoDrop1000

Genomic DNA Isolation – Geoduck Adductor Muscle & Foot

Isolated gDNA from Panopea generosa (geoduck) adductor muscle & foot samples collected by Brent & Steven on 20150811 using the DNAzol (Molecular Research Center) according to the manufacturer’s protocol, with the following adjustments:

  • 102.5mg of adductor muscle 1
  • 76.7mg of adductor muscle 2
  • 84.2mg of foot 1
  • 54.5mg of foot 2
  • Tissues homogenized in 750μL of DNAzol with disposable mortar/pestle tubes using 10 pestle strokes
  • After homogenization, topped off tubes to 1000μL with DNAzol and incubated @ RT for 10mins.
  • Performed optional centrifugation step (10,000g, 10mins @ RT)
  • Initial pellet wash was performed using a 70%/30% DNAzol/EtOH
  • Pellets were resuspended in 200μL of Buffer EB (Qiagen)
  • Insoluble material was pelleted (12,000g, 10mins @ RT) and supe transferred to new tubes

Spec’d on Roberts Lab NanoDrop1000 (ThermoFisher) and stored temporarily at 4C to avoid freeze-thawing before sending off for sequencing next week.

 

Results:

 

 

There was a great deal of insoluble material from the get-go that was carried through the entire isolation.

Overall, the 260/280 ratios look pretty good, but the 260/230 ratios are just trash. As can be seen in the plots above, there is clearly significant absorbance in the 230 – 250nm, suggesting some contaminant carryover (phenol/salt).

Will evaluate gDNA integrity on agarose gel.

Yields from this isolation:

Adductor muscle 1: 11.03μg

Adductor muscle 2: 1.95μg

Foot 1: 4.6μg

Foot 2: 1.64μg

 

Total geoduck gDNA from this isolation: 19.2μg

 

Total geoduck gDNA accumulated for this project: 69μg

Still need an additional 4μg at a minimum! Will isolate more gDNA tomorrow…

Genomic DNA Isolation – Olympia oyster adductor musle & mantle

Previously isolated gDNA from these tissues on 20150901. However, found out after the isolations that BGI needs >73μg of gDNA for the genome sequencing project, which is significantly more than I obtained previously.

Isolated gDNA from Ostrea lurida (Olympia oyster) adductor muscle & mantle samples collected by Brent & Steven on 20150812 using DNAzol (Molecular Research Center) according to the manufacturer’s protocol, with the following adjustments:

  • 89.8mg of adductor muscle
  • 92.2mg of mantle
  • Tissues homogenized in 750μL of DNAzol with disposable mortar/pestle tubes using 10 pestle strokes
  • After homogenization, topped off tubes to 1000μL with DNAzol and incubated @ RT for 10mins.
  • Performed optional centrifugation step (10,000g, 10mins @ RT)
  • Initial pellet wash was performed using a 70%/30% DNAzol/EtOH
  • Pellets were resuspended in 200μL of Buffer EB (Qiagen)
  • Insoluble material was pelleted (12,000g, 10mins @ RT) and supe transferred to new tubes

Spec’d on Roberts Lab NanoDrop1000 (ThermoFisher) and stored temporarily at 4C to avoid freeze-thawing before sending off for sequencing.

 

Results:

 

There was a great deal of insoluble material from the get-go that was carried through the entire isolation.

Overall, the 260/280 ratios look pretty good, but the 260/230 ratios are just trash. As can be seen in the plots above, there is clearly significant absorbance in the 230 – 250nm, suggesting some contaminant carryover (phenol/salt).

Will evaluate gDNA integrity on agarose gel.

Total yield from this isolation is still below the minimum quantity of gDNA needed for the sequencing project. Will need to perform another round of gDNA isolation.

Genomic DNA Isolation – Geoduck Adductor Muscle & Foot

Previously isolated gDNA from these tissues on 20150828. However, found out after the isolations that BGI needs >73μg of gDNA for the genome sequencing project, which is significantly more than I obtained previously.

Isolated gDNA from Panopea generosa (geoduck) adductor muscle & foot samples collected by Brent & Steven on 20150811 using the DNAzol (Molecular Research Center) according to the manufacturer’s protocol, with the following adjustments:

  • 58.8mg of adductor muscle 1
  • 84.0mg of adductor muscle 2
  • 70.3mg of foot 1
  • 95.1mg of foot 2
  • Tissues homogenized in 750μL of DNAzol with disposable mortar/pestle tubes using 10 pestle strokes
  • After homogenization, topped off tubes to 1000μL with DNAzol and incubated @ RT for 10mins.
  • Performed optional centrifugation step (10,000g, 10mins @ RT)
  • Initial pellet wash was performed using a 70%/30% DNAzol/EtOH
  • Pellets were resuspended in 200μL of Buffer EB (Qiagen)
  • Insoluble material was pelleted (12,000g, 10mins @ RT) and supe transferred to new tubes

Spec’d on Roberts Lab NanoDrop1000 (ThermoFisher) and stored temporarily at 4C to avoid freeze-thawing before sending off for sequencing next week.

 

Results:

 

There was a great deal of insoluble material from the get-go that was carried through the entire isolation.

Overall, the 260/280 ratios look pretty good, but the 260/230 ratios are just trash. As can be seen in the plots above, there is clearly significant absorbance in the 230 – 250nm, suggesting some contaminant carryover (phenol/salt). Oddly, the side-by-side isolations from two different collections of the same tissue type yielded drastically different quantities of gDNA than each other.

Will evaluate gDNA integrity on agarose gel.

Total yield from this isolation is still far below the minimum quantity of gDNA needed for the sequencing project. Will need to perform another round of gDNA isolation.

Genomic DNA Isolation – Olympia oyster adductor musle & mantle

Isolated gDNA from Ostrea lurida (Olympia oyster) adductor muscle & mantle samples collected by Brent & Steven on 20150812 using the E.Z.N.A Mollusc DNA Kit (Omega Bio-Tek) according to the manufacturer’s protocol, with the following adjustments:

  • 29.8mg of adductor muscle
  • 28.7mg of mantle
  • Tissues homogenized in 350μL of ML1 Buffer with disposable mortar/pestle tubes using only three pestles strokes
  • Homogenized tissue incubated in ML1 Buffer + Proteinase K @ 60C for 2.5hrs
  • Added 310μL of MBL Buffer to adductor muscle sample and 265μL of MBL Buffer to mantle sample
  • Added 620μL of 100% EtOH and 530μL of 100% EtOH to the adductor muscle and mantle sample, respectively.
  • Eluted with 50μL Elution Buffer.

Spec’d on Roberts Lab NanoDrop1000 (ThermoFisher) and stored temporarily at 4C to avoid freeze-thawing before sending off for sequencing.

 

Results:

 

 

 

Yields are good (~6μg for the adductor muscle and ~10μg for the mantle).

The adductor muscle sample looks pretty good (perfect 260/280 ratio and solid 260/230 ratio), while the mantle sample looks OK (good 260/280 ratio, tolerable 260/230 ratio). Will run samples on gel to assess gDNA integrity.

 

Genomic DNA Isolation – Geoduck Adductor Muscle & Foot

Isolated gDNA from Panopea generosa (geoduck) adductor muscle & foot samples collected by Brent & Steven on 20150811 using the E.Z.N.A Mollusc DNA Kit (Omega Bio-Tek) according to the manufacturer’s protocol, with the following adjustments:

  • 41.8mg of adductor muscle
  • 30.0mg of foot used
  • Tissues homogenized in 350μL of ML1 Buffer with disposable mortar/pestle tubes using only three pestles strokes
  • Homogenized tissue incubated in ML1 Buffer + Proteinase K @ 60C for 2.5hrs
  • Added 265μL of MBL Buffer
  • Added 514μL of 100% EtOH.
  • Eluted with 75μL Elution Buffer.

Spec’d on Roberts Lab NanoDrop1000 (ThermoFisher) and stored temporarily at 4C to avoid freeze-thawing before sending off for sequencing next week.

 

Results:

 

 

 

Yields are good (~7ug).

Quality (260/280 ratios) looks great for both samples.

260/230 ratio not very good for adductor muscle, but perfect for foot tissue.

Will run samples on gel to assess gDNA integrity.

 

RNA Quantification – O.lurida 1hr post-mechanical heat stress DNased RNA

DNased RNA from 07272015 was quantified using the Roberts Lab NanoDrop1000.

 

Results:

The 260/280 ratios don’t look great, but that is most likely due to the DNase treatment. The DNase that’s added to each sample isn’t actually removed, so that additional protein will skew the 260/280 ratios. Will proceed with qPCR to check for any residual gDNA in these samples.

 

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

 

Bioanalyzer – Geoduck Gonad RNA Quality Assessment

Before proceeding with transcriptomics for this project, we need to assess the integrity of the RNA via Bioanalyzer.

RNA that was previously isolated on 20150508, 20150505, 20150427, and 20150424 (those notebook entries have been updated to report this consolidation and have a link to this notebook entry) were consolidated into single samples (if there had been multiple isolations of the same sample) and spec’d on the Roberts Lab NanoDrop1000:

Google Sheet: 20150528_geoduck_histo_RNA_ODs

NOTE: Screwed up consolidation of Geoduck Block 03 sample (added one of the 04 dupes to the tube, so discarded 03).

RNA was stored in Shellfish RNA Box #5.

RNA was submitted to to Jesse Tsai at University of Washington Department of Environmental and Occupational Health Science Functional Genomics Laboratory for running on the Agilent Bioanalyzer 2100, using either the RNA Pico or RNA Nano chips, depending on RNA concentration (Pico for lower concentrations and Nano for higher concentrations – left decision up to Jesse).

 

Results:

Bioanalzyer 2100 Pico Data File (XAD): SamWhite_Eukaryote Total RNA Pico_2015-05-28_12-50-00.xad
Bioanalzyer 2100 Nano Data File (XAD): SamWhite_Eukaryote Total RNA Nano_2015-05-28_13-22-53.xad

 

Pico Gel Representation

 

Pico Electropherogram

 

Nano Gel Representation

 

Nano Electropherogram

 

Jesse alerted me to the fact that they did not have any ladder to use on the Nano chip, as someone had used the remainder, but failed to order more. I OK’d him to go ahead with the Nano chip despite lacking ladder, as we primarily needed to assess RNA integrity.

 

Bad Samples:

  • Geo 04 – No RNA detected
  • Geo 65, 67, 68 – These three samples show complete degradation of the RNA (i.e. no ribosomal band present, significant smearing on the gel representation).

All other samples look solid. Will discuss with Steven and Brent on how they want to proceed.

Full list of samples for this project (including the Block 03 sample not included in this analysis; see above). Grace’s notebook will have details on what the numbering indicates (e.g. developmental stage).

  • block 02
  • block 03 (no RNA)
  • block 04 (no RNA)
  • block 07
  • block 08
  • block 09
  • block 34
  • block 35
  • block 38
  • block 41
  • block 42
  • block 46
  • block 51
  • block 65 (degraded RNA)
  • block 67 (degraded RNA)
  • block 68 (degraded RNA)
  • block 69
  • block 70

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.