Tag Archives: gel

DNA Isolation – Geoduck gDNA for Illumina-initiated Sequencing Project

We were previously approached by Cindy Lawley (Illumina Market Development) for possible participation in an Illumina product development project, in which they wanted to have some geoduck tissue and DNA on-hand in case Illumina green-lighted the use of geoduck for testing out the new sequencing platform on non-model organisms. Well, guess what, Illumina has give the green light for sequencing our geoduck! However, they need at least 4μg of gDNA, so I’m isolating more.

Isolated DNA from ctenidia tissue from the same Panopea generosa individual used for the BGI sequencing efforts. Tissue was collected by Brent & Steven on 20150811.

Used the E.Z.N.A. Mollusc Kit (Omega) to isolate DNA from five separate ~60mg pieces of ctenidia tissue according to the manufacturer’s protocol, with the following changes:

  • Samples were homogenized with plastic, disposable pestle in 350μL of ML1 Buffer
  • Incubated homogenate at 60C for 1hr
  • No optional steps were used
  • Performed three rounds of 24:1 chloroform:IAA treatment
  • Eluted each in 50μL of Elution Buffer and pooled into a single sample

Quantified the DNA using the Qubit dsDNA BR Kit (Invitrogen). Used 1μL of DNA sample.

Concentration = 162ng/μL (Quant data is here [Google Sheet]: 20170105_gDNA_geoduck_qubit_quant

Yield is great (total = ~32μg).

Evaluated gDNA quality (i.e. integrity) by running 162ng (1μL) of sample on 0.8% agarose, low-TAE gel stained with ethidium bromide.

Used 5μL of O’GeneRuler DNA Ladder Mix (ThermoFisher).

 

Results:

 

 

DNA looks good: bright high molecular weight band, minimal smearing, and minimal RNA carryover (seen as more intense “smear” at ~500bp).

Will send off 10μg (they only requested 4μg) so that they have extra to work with in case they come across any issues.

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DNA Isolation – Geoduck gDNA for Potential Illumina-initiated Sequencing Project

We were approached by Cindy Lawley (Illumina Market Development) yesterday to see if we’d be able to participate in some product development. We agreed and need some geoduck DNA to send them, in case she’s able to get our species greenlighted for use.

Isolated DNA from ctenidia tissue from the same Panopea generosa individual used for the BGI sequencing efforts. Tissue was collected by Brent & Steven on 20150811.

Used the E.Z.N.A. Mollusc Kit (Omega) to isolate DNA from two separate 50mg pieces of ctenidia tissue according to the manufacturer’s protocol, with the following changes:

  • Samples were homogenized with plastic, disposable pestle in 350μL of ML1 Buffer
  • Incubated homogenate at 60C for 1hr
  • No optional steps were used
  • Performed three rounds of 24:1 chloroform:IAA treatment
  • Eluted each in 50μL of Elution Buffer and pooled into a single sample

Quantified the DNA using the Qubit dsDNA BR Kit (Invitrogen). Used 1μL of DNA sample.

Concentration = 19.4ng/μL (Quant data is here [Google Sheet]: 20161221_gDNA_qubit_quant

Yield is low (~1.8μg), but have enough to satisfy the minimum of 1μg requested by Cindy Lawley.

Evaluated gDNA quality (i.e. integrity) by running ~250ng (12.5μL) of sample on 0.8% agarose, low-TAE gel stained with ethidium bromide.

Used 5μL of O’GeneRuler DNA Ladder Mix (ThermoFisher).

 

Results:

 

 

 

 

Overall, the sample looks good. Strong, high molecular weight band is present with minimal smearing. However, there is a smear in the ~500bp range. This is most likely residual RNA. This is surprsing since the E.Z.N.A Mollusc Kit includes n RNase step. Regardless, having intact, high molecular weight DNA is the important part for this project. Will prepare to send remainder (~1.5μg) of geoduck to Illumina with other requested samples.

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DNA Isolation – Ostrea lurida DNA for PacBio Sequencing

In an attempt to improve upon the partial genome assembly we received from BGI, we will be sending DNA to the UW PacBio core facility for additional sequencing.

Isolated DNA from mantle tissue from the same Ostrea lurida individual used for the BGI sequencing efforts. Tissue was collected by Brent & Steven on 20150812.

Used the E.Z.N.A. Mollusc Kit (Omega) to isolate DNA from two separate 50mg pieces of mantle tissue according to the manufacturer’s protocol, with the following changes:

  • Samples were homogenized with plastic, disposable pestle in 350μL of ML1 Buffer
  • Incubated homogenate at 60C for 1.5hrs
  • No optional steps were used
  • Performed three rounds of 24:1 chloroform:IAA treatment
  • Eluted each in 50μL of Elution Buffer and pooled into a single sample

Quantified the DNA using the Qubit dsDNA BR Kit (Invitrogen). Used 1μL of DNA sample.

Concentration = 326ng/μL (Quant data is here [Google Sheet]: 20161214_gDNA_Olurida_qubit_quant

Yield is good and we have more than enough (~5μg is required for sequencing) to proceed with sequencing.

Evaluated gDNA quality (i.e. integrity) by running ~500ng (1.5μL) of sample on 0.8% agarose, low-TAE gel stained with ethidium bromide.

Used 5μL of O’GeneRuler DNA Ladder Mix (ThermoFisher).

Results:

 

 

Overall, the gel looks OK. A fair amount of smearing, but a strong, high molecular weight band is present. The intensity of the smearing is likely due to the fact that the gel is overloaded for this particular well size. If I had used a broader comb and/or loaded less DNA, the band would be more defined and the smearing would be less prominent.

Will submit sample to the UW PacBio facility tomorrow!

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Agarose Gel – Oly gDNA for BS-seq Libraries, Take Two

The gel I ran earlier today looked real rough, due to the fact that I didn’t bother to equalize loading quantities of samples (I just loaded 1μL of all samples regardless of concentration). So, I’m repeating it using 100ng of DNA from all samples.

Additionally, this gel also includes C.gigas samples that Katie Lotterhos sent to us to see how they look.

Ran a 0.8% agarose, low-TAE gel, stained with ethidium bromide.

Results:

 

Look at that! The samples look MUCH nicer when they’re not overloaded and uniformly loaded!

Most have a prominent high molecular weight band (the band that’s closes to the top of the ladder, not the DNA visible in the wells). All exhibit smearing, but 2NF1 shows a weird accumulation of low molecular weight DNA.

Katie’s C.gigas samples (M1, M2, M3) look similar to the Olympia oyster gDNA, however her samples appear to have residual RNA in them (the fuzzy band ~500bp).

Will discuss with Steven which samples he wants to use for bisulfite treament and library construction.

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Agarose Gel – Oly gDNA for BS-seq Libraries

Ran 1μL of each sample from yesterday’s DNA isolation on a 0.8% agarose, low-TAE gel, stained with ethidium bromide.

 

Results:

 

 

Since I didn’t load equal quantities of DNA, the intensities across the various samples is highly variable.

Those samples with high degree of smearing are also those with the highest concentrations. Thus, one would expect to be able to visualize a greater range of DNA sizes in a gel (because more DNA is present). Notice the samples with nice, high molecular weight bands and little smearing (1NF16, 1NF17). These are less than half the concentrations of all the samples that exhibit extensive smearing (2NF3, 2NF8, 1NF12). So, I think all samples will be fine for proceeding with bisulfite conversion and subsequent library construction.

However, I should re-run this gel using equalized DNA quantities for all samples…

 

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DNA Quality Assessment – Geoduck & Olympia Oyster gDNA

Have three separate sets of geoduck & olympia oyster gDNA that need to be run on gels before sending to BGI for genome sequencing:

GEODUCK

 

OLYMPIA OYSTER

 

Ran 100ng of each sample on a 0.8% agarose 1x modified TAE gel w/EtBr.

Results:

 

All the samples from both sets appear to be overloaded. Overloading is generally seen as the streaking seen immediately above each band.

GEODUCK

Overall, the samples look pretty good. Sadly, the worst of the three (due to the most smearing – i.e. degradation) appears to be the DNA extracted using the E.Z.N.A. Mollusc Kit (Omega BioTek).

Also of note are the two bands present in the DNAzol sample. These bands are likely ribosomal RNA because I neglected to perform a RNase treatment during the extraction. Doh!

 

OLYMPIA OYSTER

None of them are particularly great. Just like the geoduck set, the worst of the three came from the E.Z.N.A Mollusc Kit (Omega BioTek).

Also, just like the geoduck set, there are two bands present in the DNAzol sample. These bands are likely ribosomal RNA because I neglected to perform a RNase treatment during the extraction. Doh!

The phenol-chloroform clean up sample is either jacked up or severely overloaded, based on the crazy streaking that’s present. However, this sample looked similar after the initial extraction on 20151113.

 

I will send these samples separately (i.e. will not pool them into single samples) to BGI to run QC and, hopefully, add them to the DNA they already have to complete the genome sequencing for these two projects.

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DNA Quality Assessment – Geoduck, Oly & Oly 2SN

I recently ran gDNA isolated for geoduck and Olympia oyster genome sequencing, as well as gDNA isolated from the Olympia oyster reciprocal transplant experiment out on a Bioanalyzer (Agilent) using the DNA 12000 chips. The results from the chip were a bit confusing and difficult to assess exactly what was going on with the DNA.

So, I ran 5μL of each of those samples on a 0.8% agarose 1x modified TAE gel w/EtBr to get a better look at how the samples actually looked.

Results:

 

Both the geoduck and the Olympia oyster samples for genome sequencing show intact, high molecular weight bands with some smearing (i.e. degradation). The oly sample looks a bit funky, most likely due to a gel anomaly. I’ll quantify these using a dye-based method for a more accurate quantification before sending off to BGI.

The Fidalgo 2SN samples all have intact, high molecular weight bands, but most of the samples show extensive smearing (i.e. degradation). However, sample 2SN 35 has no visible DNA at all.

Here’s a table highlighting the differences between the Fidalgo gDNA samples:

Sample Fresh/Frozen Isolator
10 Fresh Sam
11 Fresh Sam
12 Fresh Sam
20 Fresh Mrunmayee
21 Fresh Mrunmayee
22 Fresh Mrunmayee
32 Frozen Sam
33 Frozen Sam
34 Frozen Sam
35 Frozen Sam

 

The fresh ctenidia samples were isolated by me on 20151021 and by Mrunmayee on 20151023. The frozen ctenidia samples were isolated by me on 20151103.

It’s interesting to note that Mrunmayee’s isolations appear to exhibit the least amount of degradation. Besides her handling the samples, the primary difference is that her samples were incubated in the buffer/Pro K solution O/N @ 37C, while my fresh samples were incubated @ 60C for 3hrs and my frozen samples were incubated @ 60C for 1hr. Overall, though, the frozen samples look the worst.

Finally, it’s also interesting to see that the two samples isolated using DNazol (geoduck and Olympia oyster genome samples) migrate more slowly than the remaining Olympia oyster samples which were isolated with the E.Z.N.A. Mollusc Kit.

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PCR – Oly RAD-seq Prep Scale PCR

Continuing with the RAD-seq library prep. Following the Meyer Lab 2bRAD protocol.
After determining the minimum number of PCR cycles to run to generate a visible, 166bp band on a gel yesterday, ran a full library “prep scale” PCR.

 

REAGENT SINGLE REACTION (μL) x11
Template 40 NA
ILL-HT1 (1μM) 5 55
ILL-BC# (1μM) 5 NA
NanoPure H2O 5 55
dNTPs (1mM) 20 220
ILL-LIB1 (10μM) 2 22
ILL-LIB2 (10μM) 2 22
5x Q5 Reaction Buffer 20 220
Q5 DNA Polymerase 1 11
TOTAL 100 550

 

Combined the following for PCR reactions:

  • 55μL PCR master mix
  • 40μL ligation mix
  • 5μL of ILL-BC# (1μM) – The barcode number and the respective sample are listed below.

 

SAMPLE BARCODE SEQUENCE
Oly RAD 02  1  CGTGAT
Oly RAD 03  2  ACATCG
Oly RAD 04  3  GCCTAA
Oly RAD 06  4  TGGTCA
Oly RAD 07  5  CACTGT
Oly RAD 08  6  ATTGGC
Oly RAD 14  7  GATCTG
Oly RAD 17  8  TCAAGT
Oly RAD 23  9  CTGATC
Oly RAD 30 10 AAGCTA

 

Cycling was performed on a PTC-200 (MJ Research) with a heated lid:

STEP TEMP (C) TIME (s)
Initial Denaturation
  • 98
  • 30
17 cycles
  • 98
  • 60
  • 72
  • 5
  • 20
  • 10

 

After cycling, added 16μL of 6x loading dye to each sample.

Loaded 10μL of ladder on each of the two gels.

Results:

 

Things looked fine. Excised the bands from each sample indicated by the green arrow. Before and after gel images show regions excised. Will purify the bands and quantify library yields.

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PCR – Oly RAD-seq Test-scale PCR

Continuing with the RAD-seq library prep. Following the Meyer Lab 2bRAD protocol.

Prior to generating full-blown libraries, we needed to run a “test-scale” PCR to identify the minimum number of cycles needed to produce the intended product size (166bp).

I ran PCR reactions on a subset (Sample #: 2, 3, 17, & 30) of the 10 samples that I performed adaptor ligations on 20151029.

PCR reactions were set up on ice in 0.5mL PCR tubes.

REAGENT SINGLE REACTION (μL) x4.4
Template 8 NA
NanoPure H2O 1 4.4
dNTPs (1mM) 4 17.6
ILL-LIB1 (10μM) 0.4 1.76
ILL-LIB2 (10μM) 0.4 1.76
ILL-HT1 (1μM) 1 4.4
ILL-BC1 (1μM) 1 4.4
5x Q5 Reaction Buffer 4 17.6
Q5 DNA Polymerase 0.2 0.88
TOTAL 20 52.8

 

Combined 12μL of master mix with 8μL of the ligation reaction from earlier today.

Cycling was performed on a PTC-200 (MJ Research) with a heated lid:

STEP TEMP (C) TIME (s)
Initial Denaturation
  • 98
  • 30
27 cycles
  • 98
  • 60
  • 72
  • 5
  • 20
  • 10

We’re following the “1/4 reduced representation” aspect of the protocol. As such, 5μL of each reaction was pulled immediately after the extension (72C – machine was paused) of cycles 12, 17, 22, & 27 in order to determine the ideal number of cycles to use. Also ran the ligation reactions (labeled “Ligations” on the gel below) of the samples as a pre-PCR comparison. Treated them the same as the PCR reactions: mixed 8μL of the ligation with 12μL of H2O, used 5μL of that mix to load on gel.

These samples were run on a 1x modified TAE 1.2% agarose gel (w/EtBr).

 

Results:

Gel image denoting sample numbers within each cycle number. Green arrow indicates the expected migration of our target band size of 166bp.

Looks like cycle 17 is the minimum cycle number with which we begin to see a consistent ~166bp band. Will continue on with the “prep-scale” PCR using 17 cycles.

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PCR – RLOv In-situ Hybridization (ISH) Probes

Ran probe-labeling PCRs to use in in-situ hybridization (ISH) using the PCR DIG Probe Sysnthesis Kit (Roche). Generated PCR probes for using the following BamHI-linearized plasmids:

  • pCR2.1/RLOv_membrane_gene_1
  • pCR2.1/RLOv_membrane_gene_1
  • pCR2.1/RLOv_tail_fiber

The Roche protocol recommends using only 10pg of plasmid DNA for probe labelling. As such, all three probes were diluted 1:10,000. A 1:1000 (999μL H2O + 1μL of plasmid) was made first. Then a 1:10 dilution was made (90μL H2O + 10μL from 1:1000 dilution of plasmid).

Additionally, I ran half reactions to conserve kit components. Roche recommends 50μL reactions; I ran 25μL and scaled all components appropriately.

All reactions were set up on ice and run in 0.2mL strip-cap PCR tubes.

Reaction calculations are here (Google Sheet): 20151109 – RLOv ISH Probe PCRs

Cycling params:

  1. 95C – 5mins
  2. 95C – 15s
  3. 55C – 15s
  4. 72C – 30s
  5. Go to Step 2, repeat 39 times.
  6. 72C – 10mins

After the PCR, 5μL of each reaction was run on a gel.

Results:

Hyperladder I (Bioline)

PCR DIG probe labelling products run on 1.1% agarose 1x TBE gel stained w/EtBr. A ‘+’ indicates DIG reaction, while a ‘-‘ indicates no DIG in reaction.

Two reactions were run for each plasmid: one with the DIG label (indicated by a ‘+’) and one without (indicated by a ‘-‘). If the labeling was successful, the PCR products from those reactions containing DIG will be larger (i.e. migrate slower) than those without. That is exactly what we see in each of the three potential ISH targets.

So, we now have three ISH probes ready for action! Will proceed with making fresh ISH buffers and ISH.

Probes were transferred to 0.5mL snap cap tubes and stored in my -20C box.

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