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 @ 95^oC 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.

Isolated DNA from two of the geoduck hatchery metagenome samples Emma delivered on 20180313 to get an idea of what type of yields we might get from these.

• MG 5/15 #8
• MG 5/19 #6

As mentioned in my notebook entry upon receipt of these samples, I’m a bit skeptical will get any sort of recovery, based on sample preservation.

Isolated DNA using DNAzol (MRC, Inc.) in the following manner:

1. Added 1mL of DNAzol to each sample; mixed by pipetting.
2. Added 0.5mL of 100% ethanol; mixed by inversion.
3. Pelleted DNA 5,000g x 5mins @ RT.
4. Discarded supernatants.
5. Wash pellets (not visible) with 1mL 75% ethanol by dribbling down side of tubes.
6. Pelleted DNA 5,000g x 5mins @ RT.
7. Discarded supernatants and dried pellets for 5mins.
8. Resuspended DNA in 20uL of Buffer EB (Qiagen).

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

5uL of each sample were used.

Results:

As expected, both samples did not yield any detectable DNA.

Will discuss with Steven on what should be done with the remaining samples.

Finished the ethanol precipitation as described in the MethylMiner (Invitrogen) manual which Yaamini had previously initiated: https://yaaminiv.github.io/Virginica-MBDSeq-Day4/

Samples were resuspended in 25μL of Buffer EB (Qiagen) and transferred to 0.5mL snap cap tubes. All tubes were labeled as: MBD CV #

Quantified the Crassostrea virginica MBD-enriched DNA with the Qubit 3.0 (ThermoFisher) and the Qubit dsDNA High Sensitivity (HS) Kit (ThermoFisher).

Used 1uL of template DNA.

Results:

Quantification Spreadsheet (Google Sheet):20180207_qubit_DNA_HS_MBD_virginica

One sample (MBD CV 106) may not be usable due to low yield. However, the remainder should work fine.

I’ve sent them all to ZymoResearch for bisulfite treatment, library construction, and Illumina sequencing.

FedEx tracking: 771429590026

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

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.