Genomic DNA Isolation – Geoduck Adductor Muscle & Foot

Previously isolated gDNA from these tissues on 20150828. However, found out after the isolations that BGI needs >73μg of gDNA for the genome sequencing project, which is significantly more than I obtained previously.

Isolated gDNA from Panopea generosa (geoduck) adductor muscle & foot samples collected by Brent & Steven on 20150811 using the DNAzol (Molecular Research Center) according to the manufacturer’s protocol, with the following adjustments:

  • 58.8mg of adductor muscle 1
  • 84.0mg of adductor muscle 2
  • 70.3mg of foot 1
  • 95.1mg of foot 2
  • Tissues homogenized in 750μL of DNAzol with disposable mortar/pestle tubes using 10 pestle strokes
  • After homogenization, topped off tubes to 1000μL with DNAzol and incubated @ RT for 10mins.
  • Performed optional centrifugation step (10,000g, 10mins @ RT)
  • Initial pellet wash was performed using a 70%/30% DNAzol/EtOH
  • Pellets were resuspended in 200μL of Buffer EB (Qiagen)
  • Insoluble material was pelleted (12,000g, 10mins @ RT) and supe transferred to new tubes

Spec’d on Roberts Lab NanoDrop1000 (ThermoFisher) and stored temporarily at 4C to avoid freeze-thawing before sending off for sequencing next week.

 

Results:

 

There was a great deal of insoluble material from the get-go that was carried through the entire isolation.

Overall, the 260/280 ratios look pretty good, but the 260/230 ratios are just trash. As can be seen in the plots above, there is clearly significant absorbance in the 230 – 250nm, suggesting some contaminant carryover (phenol/salt). Oddly, the side-by-side isolations from two different collections of the same tissue type yielded drastically different quantities of gDNA than each other.

Will evaluate gDNA integrity on agarose gel.

Total yield from this isolation is still far below the minimum quantity of gDNA needed for the sequencing project. Will need to perform another round of gDNA isolation.

Agarose Gel – Geoduck & Olympia Oyster gDNA Integrity Check

Ran 0.8% agarose 1x modified TAE gel stained with EtBr to assess the integrity of geoduck gDNA (from 20150828) and Olympia oyster gDNA (from 20150901).

Ran 500ng of each sample:

Geoduck adductor muscle: 4.87μL

Geoduck foot: 4.50μL

Oly adductor muscle: 4.22μL

Oly mantle: 2.51μL

Results:

 

 

The gel was loaded in the same order as the sample volumes listed above.

The gel is a bit disappointing.

Geoduck adductor

High molecular weight band present, but extremely faint. Not too much smearing, but accumulation of low molecular weight smudge suggests residual RNA. This is despite the fact that the kit used for isolation (E.Z.N.A. Mollusc DNA Kit) has a RNase treatment. This residual RNA could explain why the amount loaded on the gel appears to be so little compared to other samples (the RNA is contributing to the absorbance at 260nm, thus inflating the calculated concentration of gDNA).

Geoduck foot

High molecular weight band present and bright. Some smearing (i.e. degradation) present, along with degarded DNA visible at ~500bp. This sample may require a Bioanalyzer run to accurately quantify the high molecular weight DNA, as the lower molecular weight DNA (i.e. degarded DNA) is “artificially” inflating the concentration of the DNA in the sample.

Oly adductor

High molecular weight band present and bright. Some smearing (i.e. degradation) present, along with degarded DNA visible at ~500bp. This sample may require a Bioanalyzer run to accurately quantify the high molecular weight DNA, as the lower molecular weight DNA (i.e. degarded DNA) is “artificially” inflating the concentration of the DNA in the sample.

Oly mantle

High molecular weight band present, but extremely faint. Some smearing and a smudge around 500bp, indicating degraded DNA. The low visibility of this sample on the gel suggests that the concentration determined by the NanoDrop1000 is inaccurate. However, unlike the geoduck adductor sample, it doesn’t appear that RNA carryover is responsible, as there is no noticeable low molecular weight (~100bp) smudge.

 

Overall, the geoduck foot and the Oly adductor samples are likely usable. Currently awaiting clarification from BGI for DNA quantity requirements for the genome sequencing of these two species.

Genomic DNA Isolation – Olympia oyster adductor musle & mantle

Isolated gDNA from Ostrea lurida (Olympia oyster) adductor muscle & mantle samples collected by Brent & Steven on 20150812 using the E.Z.N.A Mollusc DNA Kit (Omega Bio-Tek) according to the manufacturer’s protocol, with the following adjustments:

  • 29.8mg of adductor muscle
  • 28.7mg of mantle
  • Tissues homogenized in 350μL of ML1 Buffer with disposable mortar/pestle tubes using only three pestles strokes
  • Homogenized tissue incubated in ML1 Buffer + Proteinase K @ 60C for 2.5hrs
  • Added 310μL of MBL Buffer to adductor muscle sample and 265μL of MBL Buffer to mantle sample
  • Added 620μL of 100% EtOH and 530μL of 100% EtOH to the adductor muscle and mantle sample, respectively.
  • Eluted with 50μL Elution Buffer.

Spec’d on Roberts Lab NanoDrop1000 (ThermoFisher) and stored temporarily at 4C to avoid freeze-thawing before sending off for sequencing.

 

Results:

 

 

 

Yields are good (~6μg for the adductor muscle and ~10μg for the mantle).

The adductor muscle sample looks pretty good (perfect 260/280 ratio and solid 260/230 ratio), while the mantle sample looks OK (good 260/280 ratio, tolerable 260/230 ratio). Will run samples on gel to assess gDNA integrity.

 

Server Email Notifications Fix – Eagle

The system was previously set to use Steven’s Comcast SMTP server. Sending a test email from Eagle failed, indicating authentication failure. I changed this to use the University of Washington’s email server for outgoing messages. Here’s how…

In the Synology Disk Station Manager (DSM):

Control Panel > Notifications

  • Service provider: Custom SMTP Server
  • SMTP server: smtp.washington.edu
  • SMTP port: 587
  • Username: myUWnetID@uw.edu
  • Password: myUWpassword

Genomic DNA Isolation – Geoduck Adductor Muscle & Foot

Isolated gDNA from Panopea generosa (geoduck) adductor muscle & foot samples collected by Brent & Steven on 20150811 using the E.Z.N.A Mollusc DNA Kit (Omega Bio-Tek) according to the manufacturer’s protocol, with the following adjustments:

  • 41.8mg of adductor muscle
  • 30.0mg of foot used
  • Tissues homogenized in 350μL of ML1 Buffer with disposable mortar/pestle tubes using only three pestles strokes
  • Homogenized tissue incubated in ML1 Buffer + Proteinase K @ 60C for 2.5hrs
  • Added 265μL of MBL Buffer
  • Added 514μL of 100% EtOH.
  • Eluted with 75μL Elution Buffer.

Spec’d on Roberts Lab NanoDrop1000 (ThermoFisher) and stored temporarily at 4C to avoid freeze-thawing before sending off for sequencing next week.

 

Results:

 

 

 

Yields are good (~7ug).

Quality (260/280 ratios) looks great for both samples.

260/230 ratio not very good for adductor muscle, but perfect for foot tissue.

Will run samples on gel to assess gDNA integrity.

 

RAD-Seq Library Prep Reagents

A box with the above title was established in the -20C in FTR 209 containing the following:

  • Thermo Scientific AlfI: ER1801
  • NEB T4 DNA ligase, 50 μL: M0202S
  • NEB 10 mM ATP, 1 mL: P0756S
  • Promega dNTPs (10 mM each): U1511
  • NEB Q5 Taq Polymerase, 100 units: M0491S

Oligos (100μL each in TE pH=8.0; barcode sequences are in bold)

Adaptor 1
5ILL-NR: CTACACGACGCTCTTCCGATCTNR

Anti-ILL: AGATCGGAAGAGC(InvdT)

Adaptor 2
3ILL-NR: CAGACGTGTGCTCTTCCGATCTNR

ILL-Lib1: AATGATACGGCGACCACCGA

ILL-Lib2: CAAGCAGAAGACGGCATACGA

ILL-HT1: AATGATACGGCGACCACCGAGATCTACACATGCATACACTCTTTCCCTACACGACGCTCTTCCGATCT

ILL-HT2:AATGATACGGCGACCACCGAGATCTACACCGTACGACACTCTTTCCCTACACGACGCTCTTCCGATCT

ILL-BC1: CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC

Reverse Transcription – O.lurida DNased RNA 1hr post-mechanical stress

Performed reverse transcription on the Olympia oyster DNased RNA from the 1hr post-mechanical stress samples from Jake’s project. To accommodate the large numbers of anticipated genes to be targeted in subsequent qPCRs, I prepared 100μL reactions (normally, 25μL reactions are prepared) using 250ng of each DNased RNA. A 1:10 dilution of the oligo dT primers (Promega) was prepared to improve pipetting accuracy. All incubations were performed in a thermal cycler without using a heated lid.

DNased RNA was combined with NanoPure H2O and oligo dT primers in 24 wells of a PCR plate, heated @ 70C for 10mins and immediately placed on ice. After 5mins, the plate was spun 2000g @ RT for 2mins and returned to ice.

25.25μL of a master mix containing 5x M-MLV Buffer (Promega), dNTPs (10mM each; Promega), and M-MLV Reverse Transcriptase (50U/rxn; Promega) was distributed to each well and mixed via pipetting. The plate was heated @ 42C for 1hr, 95C for 3mins. The plate was spun 2000g @ RT for 2mins and then stored @ -20C.

Plate layout and all calculations can be found here (Google Sheet): 20150806_Jake_oly_mech_stress_cDNA_calcs

qPCR – Jake’s O.lurida ctenidia 1hr post-mechanical stress DNased RNA

Ran qPCR on DNased RNA from earlier today to assess whether there was any residual gDNA after the DNase treatment with Oly_Actin_F/R primers (SR IDs: 1505, 1504).

Used 1μL from all templates.

All samples were run in duplicate.

Positive control was HL1 O.lurida DNA isolated by Jake on 20150323.

Cycling params:

  • 95C – 2.5mins
  • 40 cycles of:
    • 95C – 10s
    • 60C – 20s
  • Melt curve

Master mix calcs are here: 201500806_qPCR_Oly_DNased_RNA

qPCR Plate Layout: 20150806_qPCR_plate_Jake_Oly_DNased_RNA

Results:

qPCR Data File (Opticon): 20150806_165044.tad
qPCR Report (Google Spreadsheet): 20150806_qPCR_Report_Jake_Oly_DNased_RNA

Positive control comes up around cycle ~21.

No amplification in the no template controls.

Two wells of the DNased RNA samples exhibit amplification (E3, F6), however the corresponding respective replicate does not. Will proceed with reverse transcription.

 

Amplification Plots

Positive Controls

 

Melt Curves

Positive Controls (HL1)

DNased RNA Samples

Follow the green and red lines with the vertical bars. The different colors reflect that those are two different samples. Additionally, their respective replicates do not exhibit amplification.

RNA Quantification – O.lurida 1hr post-mechanical heat stress DNased RNA

DNased RNA from 07272015 was quantified using the Roberts Lab NanoDrop1000.

 

Results:

The 260/280 ratios don’t look great, but that is most likely due to the DNase treatment. The DNase that’s added to each sample isn’t actually removed, so that additional protein will skew the 260/280 ratios. Will proceed with qPCR to check for any residual gDNA in these samples.