Tag Archives: DNA Quantification

DNA Isolation & Quantification – Geoduck larvae metagenome filter rinses

This is another attempt to isolate DNA from two more of the geoduck hatchery metagenome samples Emma delivered on 20180313.

The previous attempt, using DNAzol, did not yield any DNA.

I isolated DNA from the following two samples:

  • MG 5/19 #4
  • MG 5/26 #4

I used the DNA Stool Kit (Qiagen), following the “Stool Human DNA” protocol with the following changes:

  • Incubated @ 95oC for 5mins after initial addition of Buffer ASL. This is a lysis step that might help increase yields (see the “Stool Pathogen Detection” protocol)
  • Did not add InhibitEX Tablet. Deemed unnecessary, since these weren’t stool samples.
  • Eluted in 50μL of Buffer AE

I opted to follow the “Stool Human DNA” protocol, as it processes a larger portion of the initial sample, compared to the “Stool Pathogen Detection” protocol (600μL vs. 200μl)

Samples were quantified using the Roberts Lab Qubit 3.0 with the Qubit High Sensitivity dsDNA Kit (Invitrogen).

10μL of each sample were used.

Results:

Neither sample yielded any detectable DNA. Will discuss with Steven.

DNA Quantification – C.virginica MBD-enriched DNA

Quantified Crassostrea virginica MBD-enriched DNA from earlier today for Qiagen project.

Used the Qubit 3.0 (ThermoFisher) and the Qubit dsDNA Broad Range (BR) Kit (ThermoFisher).

Used 1uL of template DNA.

Results:

Quantification Spreadsheet (Google Sheet): 20180110_qubit_dsDNA_BR_MBD_virginica

Both samples had decent yields and have usable quantities for Qiagen (they wanted ~300ng from each sample):

virginica_MBD_01 – 18.3ng/uL (457.5ng = 5.7% methylated DNA capture)

virginica_MBD_02 – 19.6ng/uL (490ng = 6.1% methylated DNA capture)

Will store @ -20C until next week so that we’re not shipping so close to the weekend (shipping address is in Germany).

DNA Isolation & Quantification – Crassostrea virginica Mantle gDNA

DNA was isolated from a single adult Eastern oyster (Crassostrea virginica) for a pilot project with Qiagen to test their new DNA bisulfite conversion kit. The oyster was obtained yesterday afternoon (20171210) from the Taylo rShellfish Pioneer Square location. The oyster was stored @ 4C O/N.

The oyster was shucked and four pieces of upper mantle tissue (~35mg each) were snap frozen in liquid nitrogen (LN2). Tissues were pulverized under LN2 and then DNA was isolated separately from each sample using the E.Z.N.A. Mollusc DNA Kit (Omega) according to the manufcaturer’s protocol.

Samples were eluted with 100uL of Elution Buffer and were pooled into a single tube.

The gDNA was quantified using the Qubit 3.0 (Invitrogen) and Qubit dsDNA Broad Range Kit (Invitrogen), using 5uL of sample.

Results:

Qubit (Google Sheet): 20171211_qubit_virginica_DNA

Concentration is 58.4ng/uL.

That makes the total yield ~23.36ug (23360ng). This is more than enough to perform two separate MeDIP preps and two separate reduced representation digestions with MspI.

Will proceed with shearing of DNA for MeDIP.

DNA Isolation & Quantification – C. virginica Gonad gDNA

I isolated DNA from the Crassotrea virginica gonad samples sent by Katie Lotterhos using the E.Z.N.A. Mollusc Kit with the following modifications:

  • Samples were homogenized with plastic, disposable pestle in 350μL of ML1 Buffer
  • No optional steps were used
  • Eluted each in 100μL of Elution Buffer and pooled into a single sample

NOTE: Sample 034 did not process properly (no phase separation after 24:1 chlorform:IAA addition – along with suggested additions of ML1 Buffer) and was discarded.

Quantified the DNA using the Qubit dsDNA BR Kit (Invitrogen). Used 2μL of DNA sample.

Samples were stored in the same box the tissue was delivered in and stored in the same location in our -80C: rack 8, row 5, column 4.

Results:

Qubit (Google Sheet): 20171114_qubit_Cvirginica_gDNA

Ample DNA in all samples for MBDseq. (Refer to “Original Sample Conc.” column in spreadsheet.)

Will let Steven & Katie know.

DNA Quantification – Acropora cervicornis (Staghorn coral) DNA from Javier Casariego (FIU)

DNA samples received yesterday were quantified using the Roberts Lab Qubit 3.0 to improve quantification accuracy (samples provided by Javier were quantified via NanoDrop, which generally overestimates DNA concentration) prior to performing methylation assessment.

Quantification was performed using the dsDNA Broad Range Kit.

Used 1uL of each sample.

Results:

Three samples are too dilute for immediate use in the MethylFlash Methylated DNA Quantification Kit (Colorimetric) – max sample volume is 8uL. Will have to concentrate them (will likely use SpeedVac to prevent sample loss).

Values were added to the spreadsheet provided by Javier (Google Sheet): A.cervicornis_DNA_Extractions(May_2017).xlsx

Qubit output file (Google Sheet): 20170510_qubit_A_cervicornis_DNA

 

DNA Isolation – Geoduck gDNA for Illumina-initiated Sequencing Project

We were previously approached by Cindy Lawley (Illumina Market Development) for possible participation in an Illumina product development project, in which they wanted to have some geoduck tissue and DNA on-hand in case Illumina green-lighted the use of geoduck for testing out the new sequencing platform on non-model organisms. Well, guess what, Illumina has give the green light for sequencing our geoduck! However, they need at least 4μg of gDNA, so I’m isolating more.

Isolated DNA from ctenidia tissue from the same Panopea generosa individual used for the BGI sequencing efforts. Tissue was collected by Brent & Steven on 20150811.

Used the E.Z.N.A. Mollusc Kit (Omega) to isolate DNA from five separate ~60mg pieces of ctenidia tissue according to the manufacturer’s protocol, with the following changes:

  • Samples were homogenized with plastic, disposable pestle in 350μL of ML1 Buffer
  • Incubated homogenate at 60C for 1hr
  • No optional steps were used
  • Performed three rounds of 24:1 chloroform:IAA treatment
  • Eluted each in 50μL of Elution Buffer and pooled into a single sample

Quantified the DNA using the Qubit dsDNA BR Kit (Invitrogen). Used 1μL of DNA sample.

Concentration = 162ng/μL (Quant data is here [Google Sheet]: 20170105_gDNA_geoduck_qubit_quant

Yield is great (total = ~32μg).

Evaluated gDNA quality (i.e. integrity) by running 162ng (1μL) of sample on 0.8% agarose, low-TAE gel stained with ethidium bromide.

Used 5μL of O’GeneRuler DNA Ladder Mix (ThermoFisher).

 

Results:

 

 

DNA looks good: bright high molecular weight band, minimal smearing, and minimal RNA carryover (seen as more intense “smear” at ~500bp).

Will send off 10μg (they only requested 4μg) so that they have extra to work with in case they come across any issues.

DNA Isolation – Ostrea lurida DNA for PacBio Sequencing

In an attempt to improve upon the partial genome assembly we received from BGI, we will be sending DNA to the UW PacBio core facility for additional sequencing.

Isolated DNA from mantle tissue from the same Ostrea lurida individual used for the BGI sequencing efforts. Tissue was collected by Brent & Steven on 20150812.

Used the E.Z.N.A. Mollusc Kit (Omega) to isolate DNA from two separate 50mg pieces of mantle tissue according to the manufacturer’s protocol, with the following changes:

  • Samples were homogenized with plastic, disposable pestle in 350μL of ML1 Buffer
  • Incubated homogenate at 60C for 1.5hrs
  • No optional steps were used
  • Performed three rounds of 24:1 chloroform:IAA treatment
  • Eluted each in 50μL of Elution Buffer and pooled into a single sample

Quantified the DNA using the Qubit dsDNA BR Kit (Invitrogen). Used 1μL of DNA sample.

Concentration = 326ng/μL (Quant data is here [Google Sheet]: 20161214_gDNA_Olurida_qubit_quant

Yield is good and we have more than enough (~5μg is required for sequencing) to proceed with sequencing.

Evaluated gDNA quality (i.e. integrity) by running ~500ng (1.5μL) of sample on 0.8% agarose, low-TAE gel stained with ethidium bromide.

Used 5μL of O’GeneRuler DNA Ladder Mix (ThermoFisher).

Results:

 

 

Overall, the gel looks OK. A fair amount of smearing, but a strong, high molecular weight band is present. The intensity of the smearing is likely due to the fact that the gel is overloaded for this particular well size. If I had used a broader comb and/or loaded less DNA, the band would be more defined and the smearing would be less prominent.

Will submit sample to the UW PacBio facility tomorrow!

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 Quantification – BS-seq Oly/C.gigas Libraries

Re-quantified the libraries that were completed yesterday using the Qubit3.0 dsDNA HS (high sensitivity) assay because the library concentrations were too low for the normal broad range kit.

Results:

Qubit Quants and Library Normalization Calcs: 20151222_qubit_illumina_methylation_libraries

SAMPLE CONCENTRATION (ng/μL)
1NF11 2.42
1NF15 1.88
1NF16 2.74
1NF17 2.54
2NF5 2.72
2NF6 2.44
2NF7 2.38
2NF8 1.88
M2 2.18
M3 2.56
NF2_6 2.5
NF_18 2.66

 

Things look pretty good. The TruSeq DNA Methylation Library Kit (Illumina) suggests that the libraries produced should end up with concentrations >3ng/μL, but we have plenty of DNA here to make a pool for running on the HiSeq2500.