Tag Archives: Qubit dsDNA BR

DNA Quantification – Coral DNA from Jose M. Eirin-Lopez (Florida International University)

Quantified the DNA we received from Jose on 20160615 using the Qubit 3.0 Flouorometer (ThermoFisher) with the dsDNA Broad Range (BR) Kit according to the manufacturer’s protocol. Used 1μL of each sample.

Results are here (Google Sheet): Coral_DNA_QubitData_2016-06-30_08-45-56.xls

Here is a table of sample concentrations:

Sample Concentration(ng/μL)
H1 1 52.4
H1 5 34
H1 6 13
H1 8 22
H1 10 39
H1 12 52.4
H5 1 14.7
H5 5 20.8
H5 6 54
H5 8 18.4
H5 10 46.6
H5 12 29.8
H24 1 16.2
H24 5 25
H24 6 20.2
H24 8 22
H24 10 22
H24 12 30.6

 

Will proceed with DNA methylation assessment.

Illumina Methylation Library Construction – Oly/C.gigas Bisulfite-treated DNA

Took the bisulfite-treated DNA from 20151218 and made Illumina libraries using the TruSeq DNA Methylation Library Kit (Illumina).

Quantified the completed libraries using the Qubit 3.0 dsDNA BR Kit (ThermoFisher).

Evaluated the DNA with the Bioanalyzer 2100 (Agilent) using the DNA 12000 assay. Illumina recommended using the High Sensitivity assay, but we don’t have access to that so I figured I’d just give the DNA 12000 assay a go.

SampleName IndexNumber BarCode
1NF11 1 ATCACG
1NF15 2 CGATGT
1NF16 3 TTAGGC
1NF17 4 TGACCA
2NF5 5 ACAGTG
2NF6 6 GCCAAT
2NF7 7 CAGATC
2NF8 8 ACTTGA
M2 9 GATCAG
M3 10 TAGCTT
NF2_6 11 GGCTAC
NF_18 12 CTTGTA

 

Results:

Library Quantification (Google Sheet): 20151221_quantification_illumina_methylation_libraries

Test Name Concentration (ng/μL)
1NF11 Out of range
1NF15 2.14
1NF16 2.74
1NF17 2.64
2NF5 2.92
2NF6 Out of range
2NF7 2.42
2NF8 2.56
M2 Out of range
M3 2.1
NF2_6 2.38
NF2_18 Out of range

 

I used the Qubit’s BR (broad range) kit because I wasn’t sure what concentrations to expect. I need to use the high sensitivity kit to get a better evaluation of all the samples’ concentrations.

 

 

Bioanalyzer Data File (Bioanalyzer 2100): 2100_20expert_DNA_2012000_DE72902486_2015-12-21_16-58-43.xad

 

Ha! Well, looks like you definitely need to use the DNA High Sensitivty assay for the Bioanalyzer to pick up anything. Although, I guess you can see a slight hump in most of the samples at the appropriate sizes (~300bp); you just have to squint. ;)

DNA Isolation – Oly gDNA for BS-seq

Need DNA to prep our own libraries for bisulfite-treated high-throughput sequencing (BS-seq).

Isolated gDNA from the following tissue samples stored in RNAlater (tissue was not weighed) using DNAzol:

2NF1
2NF2
2NF3
2NF4
2NF5
2NF6
2NF7
2NF8
1NF11
1NF12
1NF13
1NF14
1NF15
1NF16
1NF17
1NF18

The sample coding breaks down as follows (see the project wiki for a full explanation):

2NF#

2 = Oysters outplanted in Fidalgo Bay

NF = Broodstock originated in Fidalgo Bay

# = Sample number

1NF#

1 = Oysters outplanted in Oyster Bay

NF = Broodstock originated in Fidalgo Bay

# = Sample number

 

DNA was isolated in the following manner:

  • Homogenized tissues in 500μL of DNAzol (Molecular Research Center; MRC).
  • Added additional 500μL of DNAzol.
  • Added 10μL of RNase A (10mg/mL, ThermoFisher); incubated 10mins @ RT.
  • Added 300μL of chloroform and mixed moderately fast by hand.
  • Incubated 5mins @ RT.
  • Centrifuged 12,000g, 10mins, RT.
  • Transferred aqueous phase to clean tube.
  • Added 500μL of 100% EtOH and mixed by inversion.
  • Pelleted DNA 5,000g, 5mins @ RT.
  • Performed 3 washes w/70% EtOH.
  • Dried pellet 3mins.
  • Resuspended in 100μL of Buffer EB (Qiagen).
  • Centrifuged 12,000g, 10mins, RT to pellet insoluble material.
  • Transferred supe to clean tube.

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): 20151216_Oly_gDNA_qubit_quants

SAMPLE CONCENTRATION (ng/μL)
2NF1 76.4
2NF2 175
2NF3 690
2NF4 11.7
2NF5 142
2NF6 244
2NF7 25
2NF8 456
1NF11 182
1NF12 432
1NF13 155
1NF14 21
1NF15 244
1NF16 112
1NF17 25.2
1NF18 278

 

Will run samples on gel tomorrow to evaluate gDNA integrity.

DNA Isolation – Olympia Oyster Outer Mantle gDNA

Isolated additional gDNA for the genome sequencing. 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 123mg of Ostrea lurida outer mantle collected by Brent & Steven on 20150812.

  • 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 137 27.4
NanoDrop1000 295 59.0

 

Yield is solid. 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 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