Tag Archives: DNA Isolation

DNA Isolation – Geoduck Ctenidia gDNA

Isolated additional gDNA for the genome sequencing. In an attempt to obtain better yields, I used ctenidia (instead of adductor muscle). Additionally, to try to improve the quality (260/280 & 260/230 ratios) of the gDNA, I added a chloroform step after the initial tissue homogenization.

Used 190mg of Panopea generosa ctenidia collected by Brent & Steven on 20150811.

  • Homogenized in 500μL of DNAzol.
  • Added additional 500μL of DNAzol.
  • Centrifuged 12,000g, 10mins, @ RT.
  • Split supernatant equally into two tubes.
  • Added 500μL of chloroform and mixed moderately fast by hand.
  • Centrifuged 12,000g, 10mins, RT.
  • Combined aqueous phases from both tubes in a clean tube.
  • Added 500μL of 100% EtOH and mixed by inversion.
  • Spooled precipitated gDNA and transferred to clean tube.
  • Performed 3 washes w/70% EtOH.
  • Dried pellet 3mins.
  • Resuspended in 200μL of Buffer EB (Qiagen).
  • Centrifuged 10,000g, 5mins, RT to pellet insoluble material.
  • Transferred supe to clean tube.

DNA was quantified using two methods: NanoDrop1000 & Qubit 3.0 (ThermoFisher).

For the Qubit, the samples were quantified using the Qubit dsDNA BR reagents (Invitrogen) according to the manufacturer’s protocol and used 1μL of sample for measurement.

Results:

Qubit Data (Google Sheet): 20151125_qubit_gDNA_geoduck_oly_quants

METHOD CONCENTRATION (ng/μL) TOTAL (μg)
Qubit 105 21.0
NanoDrop1000 173 34.6

 

Yield is definitely much, much better than adductor muscle! Should’ve switched to a different tissue a long time ago! We should finally have sufficient quantities of gDNA to allow for BGI to proceed with the rest of the genome sequencing! Will run sample on gel to evaluate integrity and then send off to BGI.

The NanoDrop & Qubit numbers still aren’t close (as expected).

The addition of the chloroform step definitely helped improve the 260/280 OD ratio (see below). However, the addition of that step had no noticeable impact on the 260/230 OD ratios, which is a bit disappointing.

 

NanoDrop Absorbance Values & Plots

 

DNA Isolation – Geoduck Adductor Muscle gDNA

Since we still don’t have sufficient gDNA for the full scope of the genome sequencing, I isolated more gDNA.

Isolated gDNA from 257mg adductor muscle tissue collected by Steven & Brent on 20150811.

Tissue was thoroughly minced with a clean razor blade and then processed with the E.Z.N.A. Mollusc Kit (Omega BioTek) with the following changes:

  • Doubled solution volumes for steps before sample was loaded on columns
  • Sample was split equally in two tubes prior to addition of 100% EtOH
  • All mixing was done by shaking – no vortexing! Done this way to, hopefully, maintain gDNA integrity
  • Elution volume = 50μL
  • Elution was repeated using the initial elution to maximize recovery while maintaining low sample volume.
  • The two preps were pooled – final volume = 79μL

DNA was quantified using two methods: NanoDrop1000 & QuantIT dsDNA BR Kit

For the Quant-IT kit, the samples were quantified using the QuantIT dsDNA BR Kit (Invitrogen) according to the manufacturer’s protocol.

Standards were run in triplicate, samples were run in duplicate.

96-well black (opaque) plate was used.

Fluorescence was measured on the Seeb Lab’s Victor 1420 plate reader (Perkin Elmer).

Results:

METHOD CONCENTRATION (ng/μL) VOLUME (μL) YIELD (ng)
NanoDrop1000 54.93 79 4,339
Quant-IT 34.52 79 2,727

 

The NanoDrop1000 overestimates the concentration of the sample by 1.6x!

Regardless, the yield isn’t all that great, which has generally been the case for all of the geoduck gDNA isolations I’ve performed. It would probably be prudent to try isolating gDNA from a different tissue to see if yields improve…

Will evaluate gDNA quality on a gel.

Fluorescence (Google Sheet): 20151124_geoduck_oly_gDNA_quants

 

NanoDrop1000 Measurements and Plots

DNA Isolation – Olympia Oyster Outer Mantle gDNA

Since we still don’t have sufficient gDNA for the full scope of the Olympia oyster genome sequencing, I isolated more gDNA.

Isolated gDNA from 118mg outer mantle tissue collected by Steven & Brent on 20150812.

Tissue was thoroughly minced with a clean razor blade and then processed with the E.Z.N.A. Mollusc Kit (Omega BioTek) with the following changes:

  • Doubled solution volumes for steps before sample was loaded on columns
  • Sample was split equally in two tubes prior to addition of 100% EtOH
  • All mixing was done by shaking – no vortexing! Done this way to, hopefully, maintain gDNA integrity
  • Elution volume = 50μL
  • Elution was repeated using the initial elution to maximize recovery while maintaining low sample volume.
  • The two preps were pooled – final volume = 79μL

DNA was quantified using two methods: NanoDrop1000 & QuantIT dsDNA BR Kit

For the Quant-IT kit, the samples were quantified using the QuantIT dsDNA BR Kit (Invitrogen) according to the manufacturer’s protocol.

Standards were run in triplicate, samples were run in duplicate.

96-well black (opaque) plate was used.

Fluorescence was measured on the Seeb Lab’s Victor 1420 plate reader (Perkin Elmer).

Results:

METHOD CONCENTRATION (ng/μL) VOLUME (μL) YIELD (ng)
NanoDrop1000 552.53 79 43,650
Quant-IT 219.07 79 17,307

 

The NanoDrop1000 overestimates the concentration of the sample by 2.5x!

Regardless, this is a solid yield and, when combined with the other Ostrea lurida gDNA that I cleaned up today, should push the total amount of gDNA submitted to BGI over the required threshold.

Will evaluate gDNA quality on a gel.

Fluorescence (Google Sheet): 20151124_geoduck_oly_gDNA_quants

 

NanoDrop1000 Measurements and Plots

 

DNA Isolations – Oly Fidalgo 2SN Ctenidia

Isolated DNA from 24 2SN ctenidia samples from Friday’s sampling (#32 – 55). Samples were thawed at RT.

DNA was isolated using the E.Z.N.A. Mollusc Kit (Omega BioTek) according to the manufacturer’s protocol with the following changes:

  • Samples were incubated @ 60C for only 1hr, per Steven’s recommendation (an attempt to prevent degradation)
  • No optional steps were performed
  • Used 300μL of MBL Buffer for all samples (this was more than the recovered volume of aqueous phase from each sample)
  • Single elution of 50μL

Samples were stored @ -20C in: Oly gDNA Oly Reciprocal Transplant Final Sampling Box #1.

Some notes:

  • Total time (including 1hr incubation): 4.5hrs.
  • Short incubation time did not completely digest samples
  • Partial tissue digestions led to difficulties in recovering entire aqueous phase, post chloroform treatment

 

DNA Isolation – Geoduck & Olympia Oyster

Amazingly, we need more gDNA for the two genome sequencing projects (geoduck and Olympia oyster). Used geoduck adductor muscle sample from Box 1 of the geoduck samples collected by Brent & Steven on 20150811. Used Olympia oyster ctenidia from Box 1 of adductor muscle sample collected by Brent & Steven on 20150812.

Tissues were split in approximately half, minced and transferred to tubes with 1mL of DNAzol + 50μg/mL of Proteinase K (Fermentas). Previously, I had just homogenized samples. I’m hoping that the overnight digestion with Proteinase K will help increase yields from these.

Tissue weights:

  • Geoduck adductor muscle tube 1: 292mg (gone)
  • Geoduck adductor muscle tube 2: 320mg (gone)
  • Olympia oyster ctenidia tube 1: 135mg (gone)
  • Olympia oyster ctenidia tube 2: 130mg (gone)

Samples were isolated using DNAzol (Molecular Research Center) according to the manufacturer’s protocol, with the following adjustments:

 

  • Samples were incubated O/N @ RT on a rotator.
  • After Proteinase K digestion, added 40μL RNAse A (100mg/mL) and incubated @ RT for 15mins.
  • Performed optional centrifugation step (10,000g, 10mins @ RT)
  • Initial pellet wash was performed using a 70%/30% DNAzol/EtOH
  • Pellets were resuspended Buffer EB (Qiagen)

Resuspension volume = 500μL total for each species

Samples were incubated O/N at RT to facilitate pellet resuspension.

NOTE: Geoduck “pellets” were not very DNA pellet-like. Very loose, white, and sort of disintegrate (but not dissolve in solution) when attempted to resuspend.

DNA Isolations – Fidalgo 2SN Reciprocal Transplants Final Samplings

The remaining Olympia oysters from Jake Heare’s reciprocal transplant experiment have been retrieved from field sites and are awaiting sampling. The oysters have been stored in the cold room (temp?) for 6 days so far.

Sampling scheme is as follows:

  1. Assign unique number to oysters
  2. Photograph with ruler for future shell measurements
  3. Weigh oysters
  4. Dissect ctenidia for DNA isolation
  5. Dissect & discard viscera (e.g. digestive gland and gonad)
  6. Weigh remaining body
  7. Preserve remaining body in RNAlater
  8. Weigh empty shells


Mrunmayee photographed & initiated dissections of oysters #3 – 8. I took over for oyster #9 -14.

All oyster data is here (Google Sheet): Oly reciprocal final sampling

DNA was isolated using the E.Z.N.A. Mollusc Kit (Omega Biotek) according to the manufacturer’s protocol with the following changes:

  • No optional steps were performed
  • Ctenidia tissue was lysed for 3hrs @ 60C
  • Single elution of 50μL

Samples were stored @ -20C in: Oly gDNA Oly Reciprocal Transplant Final Sampling Box #1.

gDNA Isolation – Geoduck Adductor Muscle

My isolation on Friday didn’t yield a sufficient quantity of gDNA for the additional DNA needed for the geoduck genome sequencing project. Used two adductor muscles (Box 1) samples collected by Brent & Steven on 20150811.

Tissue weights:

  • Geoduck adductor 1: 433mg (gone)
  • Geoduck adductor 2: 457.4mg (gone)

 

Samples were isolated using DNAzol (Molecular Research Center) according to the manufacturer’s protocol, with the following adjustments:

 

  • Tissues homogenized in 750μL of DNAzol with disposable mortar/pestle tubes using 10 pestle strokes
  • After homogenization, topped off tubes to 960μL with DNAzol, added 40μL RNAse A (100mg/mL) and incubated @ RT for 15mins.
  • Performed optional centrifugation step (10,000g, 10mins @ RT)
  • Initial pellet wash was performed using a 70%/30% DNAzol/EtOH
  • Pellets were resuspended Buffer EB (Qiagen)
  • Insoluble material was pelleted (12,000g, 10mins @ RT) and supe transferred to new tubes

NOTE: Both samples produced a stark white, “cottony” precipitate after the addition of the ethanol. This precipitate was transferred to a clean tube and processed in the same fashion.

 

Resuspension volumes

Adductor 1:  200μL

Adductor 2: 50μL

Adductor 1 & 2 fluff: 500μL each

 

Spec’d on Roberts Lab NanoDrop1000.

Results:

 

NOTE: The sample labeled “gDNA geoduck adductor 1″ is actually adductor 2. The sample labeled “gDNA geoduck adductor 1{1} is actually adductor 1. However, this is probably moot since these two samples will be pooled shortly.

I’m not going to speculate why there’re weird peaks at 240nm…

The two “fluff” samples aren’t good (extremely high 260/280 ratios, very low 260/230 ratios, and weird peak at 240nm). Not sure what the fluff is that precipitated out with the EtOH addition. Will discard them.

The two normal samples look fine. Will use them for pooling.

Yields

Adductor 1: 52.2μg

Adductor 2: 8.25μg

DNA Isolation – Geoduck & Olympia Oyster

Amazingly, we need more gDNA for the two genome sequencing projects (geoduck and Olympia oyster). Used geoduck “foot 1″ sample from Box 1 of the foot samples collected by Brent & Steven on 20150811. Used Olympia oyster adductor muscle from Box 1 of adductor muscle sample collected by Brent & Steven on 20150812.

Also need to evaluate DNA quality of initial broodstock samples from Jake’s Olympia oyster reciprocal transplant experiment. Used mantle samples stored in EtOH collected by Hannah (see her notebook entries on July 25 & Sept 5, 2013)

Tissue weights:

  • Geoduck foot: 108.5mg (gone)
  • Olympia oyster adductor: 258.7mg (gone)
  • Oly NF1A: 7.1mg (gone)
  • Oly SN49A: 20.8mg

 

Samples were isolated using DNAzol (Molecular Research Center) according to the manufacturer’s protocol, with the following adjustments:

 

  • Tissues homogenized in 750μL of DNAzol with disposable mortar/pestle tubes using 10 pestle strokes
  • After homogenization, topped off tubes to 960μL with DNAzol, added 40μL RNAse A (100mg/mL) and incubated @ RT for 15mins.
  • Performed optional centrifugation step (10,000g, 10mins @ RT)
  • Initial pellet wash was performed using a 70%/30% DNAzol/EtOH
  • Pellets were resuspended Buffer EB (Qiagen)
  • Insoluble material was pelleted (12,000g, 10mins @ RT) and supe transferred to new tubes

 

Genome sequencing resuspension volumes: 50μL

Oly reciprocoal resuspension volumes: 25μL

Spec’d on Roberts Lab NanoDrop1000.

Results:

 

Genome Sequencing Samples

The 260/280 ratios look fine. The 260/230 ratios look poor, as is usually the case after DNAzol isolations.

Yields:

Geoduck: 7.6μg

Oly: 16.5μg

The geoduck yield is insufficient to make up the quantity of gDNA still needed by BGI for sequencing. Will have to isolate more gDNA on Monday.

 

Reciprocal Transplant Samples

The 260/280 ratios look fine. The 260/230 ratios look poor, as is usually the case after DNAzol isolations.

Yields:

NF1A: 7,1μg

SN49A: 1.375μg

The yields are surprisingly good! Next up is to evaluate the gDNA quality on a gel to see if the samples from this experiment will be usable.

DNA Isolation – Olympia oyster whole body

Continued the extractions that Steven began yesterday and this morning using the E.Z.N.A. Mollusc DNA Kit (Omega Bio-Tek) after the RNase treatment @ 70C.

  • Samples were cooled to RT (~10mins)
  • Added 300μL of 100% EtOH to each (equivalent to the volume of aqueous phase Steven recovered from each sample)
  • Followed manufacturer’s protocol
  • Eluted all samples with 50μL of Elution Buffer
  • Spec’d on Roberts Lab NanoDrop1000

 

Results:

 

 

 

DNA looks absolutely pristine and has amazing yields.

Will check gDNA integrity via agarose gel tomorrow.

Data has been entered into the master spreadsheet for this project: Ostrea lurida Project Master Tissue Sample List

Genomic DNA Isolation – Geoduck Adductor Muscle & Foot

Just need a tad bit more gDNA for the geoduck genome sequencing project with BGI. Currently have ~69 and need a minimum of 73μg.

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:

  • 124.4mg of adductor muscle 1
  • Tissue homogenized in 750μL of DNAzol with disposable mortar/pestle tubes using 10 pestle strokes
  • After homogenization, topped off tube 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
  • Pellet was resuspended in 400μL of Buffer EB (Qiagen)
  • Insoluble material was pelleted (12,000g, 10mins @ RT) and supe transferred to new tube

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 ratio looks pretty good, but the 260/230 ratio is 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 assess gDNA integrity on a gel.

 

Yield of geoduck gDNA from this isolation: ~48μg

Total accumulated geoduck gDNA for this project: ~117μg

Great! Have sufficient gDNA to send to BGI (minimum of 73μg needed). Assuming gDNA integrity looks good on a gel, I will pool samples tomorrow, quantify the pooled gDNA and prepare for submission.