Tag Archives: NanoDrop1000

DNA Quantification & Quality Assessment – Geoduck & Oly gDNA

Quantified the following samples with the Roberts Lab NanoDrop1000 (ThermoFisher) and assessed DNA integrity on the Seeb Lab Bioanalyzer 2100 (Agilent) using the DNA 12000 chip assay:

Results:

Bioanalyzer Data File (XAD file): 2100 expert_DNA 12000_DE72902486_2015-11-04_15-06-32.xad

 

OK, there’s a LOT going on here. Will update this notebook with my thoughts sometime tomorrow…

 

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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.

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Plasmid Isolation – RLOv pCR2.1 Clones

Clone #1 was selected from each of the screened clones.

A sterile pipette tip was used to inoculate 5mL of 1x LBAmp100 in a 15mL conical tube. The tubes were incubated O/N @ 37C on a rocker.

3mL of liquid culture was used as input for plasmid isolation with the QIAprep Spin Mini Kit (Qiagen) according to the manufacturer’s protocol.

1mL of each culture was combined with 1mL of 50% sterile glycerol (25% glycerol final concentration) and stored @ -80C with existing bacterial stocks.

Plasmid DNA was eluted with 50μL of Buffer EB and quantified on the Roberts Lab NanoDrop1000 (ThermoFisher) in order to have a rough idea of concentrations to submit for Sanger sequencing. A dye-based quantification will be performed after sequencing results are back in order to obtain a more accurate assessment for use in ISH and/or qPCR standard curve creation.

Results:

Quality (260/280 & 260/230 ratios) look great and yields are more than sufficient. Will prep samples for Sanger sequencing.

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DNA Quantification – Pooled geoduck gDNA

Pooled the gDNA samples from earlier today & from 20151002.

The pooled volume = 260μL

Quantified on the Roberts Lab NanoDrop1000.

Results:

 

Besides the weird peak at 240nm, everything looks great – perfect 260/280 & 260/230 ratios.

Yield = 75.92μg

This gDNA has been RNase treated, so the concentration should be semi-accurate. Regardless, even if there’s a discrepancy of 50%, this should provide enough additional DNA for BGI to work with.

Will run on gel to evaluate integrity and then send to BGI.

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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

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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.

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Ethanol Precipitation – Geoduck & Olympia oyster gDNA

Pooled all of the geoduck gDNA from all the previous geoduck isolations for the Geoduck Genome Sequencing Project and pooled all of the Ostrea lurida gDNA from previous Ostrea lurida isolations for the Olympia Oyster Genome Sequencing Project.

Both sets of gDNA were ethanol (EtOH) precipitated. This was done for two reasons – to concentrate the samples to the minimum necessary concentration required by BGI (>119ng/μL) and to try to improve the poor 260/230 ratios (which are likely due to high salt carryover).

Precipitation was performed by consolidating each species of DNA in 15mL conicals, adding 0.1 volumes of 3M sodium acetate (pH= 5.2) and then adding 2.5 volumes of ice cold (-20C) 100% EtOH. Volumes used are below.

Sample DNA Vol (μL) 3M Sodium Acetate Vol. (μL) 100% EtOH Vol (μL) Total Vol (μL)
Geoduck 1860 186 5115 7161
Oly 780 78 2145 3003

Samples were mixed by inversion and incubated @ -80C for 3hrs.

DNA was pelleted by spinning tubes at 12,000g for 30mins @ 4C in a SL-50T (Sorval) rotor in a T21 (Sorval) table top centrifuge.

Supernatant was decanted and pellets were transferred to 1.5mL snap cap tubes.

Pellets were washed three times with 70% EtOH.

After the last wash, the supe was removed and pellets were air dried for 5mins @ RT.

In order to exceed the target concentration (>110ng/μL) needed by BGI, the pellets were resuspended in 500μL (150ng/μL) of EB Buffer (Qiagen). This is assuming each sample has at least 75μg of DNA.

Samples were spec’d on the Roberts Lab NanoDrop1000.

 

Results:

 

Concentrations are on point, the 260/280 ratios are still good and the 260/230 ratios are greatly improved. Samples are ready to send off to BGI for sequencing!

Total yields:

Geoduck: 84μg

Oly: 86μg

Will run these samples out on a gel to verify that the gDNA is still intact and didn’t get degraded during the precipitation.

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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

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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.

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

  • 73.5mg of adductor muscle
  • 146mg 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.

 

Yields from this isolation:

Adductor muscle: 18.75μg

Mantle: 15.9μg

 

Total Olympia oyster gDNA from this isolation: 34.65μg

 

Total Olympia oyster gDNA accumulated for this project: 88.75μg

 

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.

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