Tag Archives: Qubit dsDNA HS

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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DNA Quantification – Black Abalone DNA (Black Ab Exp. 2)

Lisa recently isolated DNA from the following samples:

08:13-05 (Black Ab exp 2)
08:13-18 (Black Ab exp 2)
08:13-24 (Black Ab exp 2)
08:13-25 (Black Ab exp 2)

I quantified the samples using the Roberts Lab Qubit 3.0 with the Qubit ds High Sensitivity kit. Used 1uL of each sample.

Samples were stored in designated boxes in -20C in Rm. 240.

Results:

Qubit output (Google Sheet): 20170413_DNA_quantification_qubit

 

SAMPLE CONCENTRATION (ng/uL)
08:13-05 62.4
08:13-18 0.536
08:13-24 0.454
08:13-25 8.8

NOTE: The entirety of sample 08:13-24 will be provided to Stan Langevin for high-throughput sequencing.

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DNA Quantification – Ava Withering Syndrome Transmission Study Samples

DNA samples from 20160818 (water filters) and 20160825 (feces) were quantified using the Roberts Lab Qubit 3.0 (Life Technologies) using the Qubit ds DNA HS (high sensitivity) reagents. Used 5μL of each sample.

Results:

Raw Qubit readout (Google Sheets):

 

Master spreadsheet for these, and future, samples for this project (Google Sheet): Ava WS Transmission DNA Extractions

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DNA Extraction & Quantification- Ava Withering Syndrome Transmission Study Water Filters

DNA was extracted from filters using the Qiagen DNeasy Blood & Tissue Kit (spin column protocol). Filters were incubated in 400uL of Buffer AL (twice the volume in the Qiagen protocol in order to completely coat the filters) and 50uL of Proteinase K (twice the volume in the Qiagen protocol) at 56C O/N. After incubation, 400uL (twice the volume in the Qiagen protocol) of 100% EtOH was added to each tube and vortexed thoroughly. The supernatant was transferred to the Qiagen spin columns and the Qiagen protocol was followed. Samples were eluted with 100uL of Buffer AE.

After extraction, the samples were quantified using the Roberts Lab Qubit 3.0 (Life Technologies) using the Qubit dsDNA HS reagents. Used 1μL of each sample.

The list of samples are listed below.

Results:

Concentrations are very low for both samples. This may, or may not, be expected, depending on volume of water filtered, where it was collected from, etc.

Raw Qubit Readout (Google Sheet): 20160818_DNA_quant_Qubit_Ava_abalone_WS
Master spreadsheet for these, and future, samples for this project (Google Sheet): Ava WS Transmission DNA Extractions

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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.

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