Tag Archives: DNA Quantification

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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DNA Quantification – Ava’s RLO Transmission DNA

Quantified the DNA I isolated on 20170504 and earlier today using the Roberts Lab’s Qubit 3.0 and the dsDNA Broad Range assay.

Used 1uL of each sample.

Results:

The following samples were below the level of sensitivity of the Qubit assay:

  • 15:09-142
  • 15:11-113
  • 15:11-147
  • 15:11-149

Qubit output data (Google Sheet): 20170509_Ava_RLO_quantification_qubit

An easier-to-read summary of all the samples is here (Google Sheet): 20170502_Ava_Ab_List

 

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DNA Quantification – RLO viability DNased RNA

I previously DNased RNA I isolated from water filters that were part of the RLO viability experiment that Lisa and the Capstone students are conducting. I checked for residual gDNA carryover via qPCR and all of the samples that were intended for dosing the abalone came up positive. It’s likely due to such a high quantity of algae that was co-filtered with the potential RLOs, leading to over-saturation of the RNAzol with DNA, resulting in the gDNA carryover.

In turn, I think the DNase treatment was insufficient for the quantity of carryover DNA.

I am planning on re-DNasing those samples, but want to quantify any residual DNA present to make sure that the samples aren’t still too concentrated for the DNase.

Samples were quantified using the Robert Lab Qubit 3.0 and the Qubit dsHS reagents (high sensitivity), using 1uL of sample.

Results:

Residual DNA is still present, but at levels that are well below the maximum that the DNase treatment (10ug) can handle. I will redo the DNase treatment on these samples. Spreadsheet is linked, and embedded below, with sample concentrations.

Spreadsheet (Google Sheet): 20170424_filter_rna_dna_quant

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