Tag Archives: DNA Quantification

DNA Quantification – Sea Lice DNA from 20180523

We previously received sea lice (Caligus tape) DNA from Cris Gallardo-Escarate at Universidad de Concepción.

Steven asked that I quantify and assess the DNA quality.

Ran the samples on the Roberts Lab Qubit 3.0 using the dsDNA BR assay (Invitrogen) and 1uL of template DNA.

Ran the samples on the Roberts Lab NanoDrop1000 to get 260/280 values for quality assessment using 2uL of template DNA. NanoDrop1000 was blanked with water, but I don’t know what solvent the DNA is currently resuspended in.


RESULTS

Qubit data (Google Sheet):

SAMPLE CONCENTRATION (ng/uL)
FEMALE 1 23.8
FEMALE 2 9.6

NANODROP DATA

TABLE


ABSORBANCE PLOTS


DNA looks super clean. Not sure what this DNA is intended for so can’t speculate much on what the implications might be for the concentrations on downstream usage.

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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.

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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).

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DNA Isolation & Quantification – Pinto Abalone

Isolated DNA from the following pinto abalone (Haliotis kamtschatkana) digestive gland tissues (stored in ethanol), collected by Sean Bennett as part of his Capstone project:

Accession Weight(mg)
15:30-01   194
15:30-04   67
15:31-01   34
15:31-02   107
15:31-03   83
15:31-04   80

Tissue was weighed and then DNA extracted.

DNA was extracted using the QIAmp Fast DNA Stool Mini Kit (Qiagen) following the manufacturer’s protocol with the following options:

  • Samples were briefly homogenized (due to their stiffness resulting from ethanol fixation) in the InhibitEX Buffer using disposable plastic pestles.
  • Homogenized tissue was incubated at 95C to maximize cell lysis
  • Followed “human DNA analysis” protocol for remainder of protocol (to maximize sample recovery)
  • Eluted DNA with 100μL Buffer ATE

Used the Roberts Lab Qubit 3.0 and the Qubit hsDNA Kit (high sensitivity). Used 1uL of template for all samples.

Samples were stored at -20C in FSH240 in the “Pinto Transcriptome DNA” box.

Results:

All samples have DNA.

Concentrations (Google Sheet): 20171226_qubit_DNA_pinto_ab

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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.

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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.

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DNA Quantification – Ava’s RLO Trasmission Samples

Quantified DNA extractions from Ava’s samples that I isolated earlier this month, as well as some older samples that I hadn’t quantified yet.

Used the Roberts Lab Qubit 3.0 and the Qubit dsDNA BR Kit (broad range). Used 5uL of template for the first and third groups and 1uL of template for the second group (see Results below).

All data was added to the master extraction spreadsheet (Google Sheet): ava_abalone_master_extraction_list

Results:

20171101_ava_rlo_quantification_qubit_01 (Google Sheet)

20171101_ava_rlo_quantification_qubit_02 (Google Sheet)

20171101_ava_rlo_quantification_qubit_03 (Google Sheet)

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DNA Quantification – Ava’s RLO Trasmission Samples

Quantified DNA extractions from Ava’s samples that I isolated earlier this month, as well as some older samples that I hadn’t quantified yet.

Used the Roberts Lab Qubit 3.0 and the Qubit hsDNA Kit (high sensitivity). Used 5uL of template for all samples.

All data was added to the master extraction spreadsheet (Google Sheet): ava_abalone_master_extraction_list

Results:

20171026_Ava_RLO_quantification_qubit_01 (Google Sheet)

20171026_Ava_RLO_quantification_qubit_02 (Google Sheet)

There were 65 samples with concentrations that were too high for the high sensitivity assay. Will re-quantify this samples using the broad range assay.

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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

 

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