Tag Archives: Qubit 3.0

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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RNA Isolation & Quantification – Tanner crab hemolymph

We received three Tanner crab (Chionoecetes bairdi)hemolymph samples from Pam Jensen (NOAA) yesterday. From her email to Steven:

Hi Steven,
I am sending:
tube #1 crab 3859/3656: 300 ul blood + 1300 ul RNAlater​

tube #2 crab 3665/3873: 300 ul blood + 1300 ul RNAlater
​tube #3 crab 3665/3873: 200 ul blood + 1400 ul RNAlater​

The tubes hold max of 1600 ul. Will know on Sun or Mon if either crab is infected w Hematodinium.

Tracking info to follow.
Pam

Samples were stored at 4C O/N.

Here’s what the samples looked like before processing:

The samples are extremely cloudy. I’m not sure if this is expected.

Processed samples using RNAzol RT (MRC) according to the manufacturer’s protocol for Total RNA Isolation.

Pelleted samples at 5000g for 5 mins and the samples looked like this:

Decided to pellet samples for an additional 10mins. The pellet was more compact. Transferred supernatant to clean tube, since it seemed to contain “debris” (maybe cells?). Processed pellet with RNAzol RT. Brief rundown of procedure (all steps at room temp):

  1. Transferred supe to clean tube.
  2. Added 1mL RNAzol RT to pellet and mixed by repeated pipetting (solution was cloudy and slightly viscous).
  3. Added 400uL of 0.1% DEPC-treated H2O and mixed vigorously by hand.
  4. Incubated for 10mins.
  5. Centrifuged 12,000g for 15mins.

    Samples looked like this:

    This is not normal. Usually the supernatant is the clear portion, while the blue layer is below that.
  6. Transferred 750uL of the clear portion to clean 1.7mL tube.

  7. Added equal volume of isopropanol, mixed by inversion. Appeared to be a very high amount of genomic DNA precipitation visible in the tube.
  8. Incubated for 10mins.
  9. Centrifuged 12,000g, 15mins.

    Samples looked like this:

    It appears that the nucleotides (the white interphase) are suspended on a “cushion” of higher density solution, instead of pelleted at the bottom of the tube.
  10. Removed/discarded higher density solution, leaving the white layer on the bottom of the tube.

  11. Centrifuged 12,000g, 15mins.
  12. Discarded supe.
  13. Washed pellet with 75% ethanol.
  14. Centrifuged 8,000g, 3mins.
  15. Repeated Steps 12, 13, & 14, 1x.
  16. Discarded ethanol.
  17. Resuspended RNA in 50uL 0.1% DEPC-treated H2O. Pellets did not solubilize on their own. I dispersed the pellets by repeated pipetting (P200). Remaining insoluble material was pelleted (12,000g, 30s) and supernatant was transferred to a new 1.6mL tube.

RNA was quantified using the Qubit 3.0 and the Qubit HS RNA Assay. Used 5uL of each sample.

Results:

20171107_qubit_tanner_crab_hemo (Google Sheet)

Sample ID Conc. (ng/uL) Total Yield (ng)
3859/3656 0.44 22
3665/3873 1.66 83
3665/3873 2.04 102

Interestingly, both samples from the same crab had similar/decent yields.

Samples were labeled and stored at -80C in Shellfish RNA Box #6

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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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Plasmid Isolation – pCR2.1/OsHv-1_ORF117 Miniprep

The last run at this failed, but I think that was due to old ampicillin stocks; leading to no selective pressure for transformants that actually contained plasmid.

I’ve since remedied that.

Grew up 5mL of culture from the only two transformants in 1xLB + 100ug/mL of (fresh!) ampicillin @ 37C on a rocking platform in a 15mL conical over night (~18hrs).

Isolated plasmid DNA from the entire 3mL of culture (repeated pelleting of bacteria in the same 1.5mL snap cap tube) using the QIAprep Spin Miniprep Kit, according to their protocol.

Eluted DNA with 50uL of EB Buffer.

Quantified on the Roberts Lab Qubit 3.0 using the dsDNA BR Kit (broad range) and 10uL of sample.

Results:

Quantification (Google Sheet): 20170817_quantification_oshv_orf117_plasmid

Colony 1 – 130ng/uL
Colony 2 – 148ng/uL

Yields look perfect. Will submit for sequencing at Genewiz (they need 10uL of ~50ng/uL DNA) and see what we have here…

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Plasmid Isolation – pCR2.1/OsHv-1_ORF117 Miniprep

Grew up 5mL of culture from re-streaked colony #1 in 1xLB + 100ug/mL of ampicillin @ 37C on a rocking platform in a 15mL conical over night (~18hrs).

Isolated plasmid DNA from the entire 5mL of culture (repeated pelleting of bacteria in the same 1.5mL snap cap tube) using the QIAprep Spin Miniprep Kit, according to their protocol.

Eluted DNA with 50uL of EB Buffer.

Quantified on the Roberts Lab Qubit 3.0 using the dsDNA BR Kit (broad range) and 1uL of sample.

Results:

The results are not good. Using 1uL of the sample, I received an error message that the concentration was out of range – too low!

Repeated, but used 10uL of sample. Concentration was displayed as 1.13ng/uL!!

This is insufficient yield/concentration for sequencing.

It’s possible that the kit is too old (no receipt date marked on the box…)? The reagents shouldn’t go bad, but can the columns? I feel like the resins in the columns are pretty stable, just like the various buffers.

The ridiculously low yields could also possibly indicate that the bacteria don’t actually have the plasmid, but PCRs from yesterday suggest otherwise.

Maybe the column was overloaded? I’ll repeat this next week, but using smaller culture size and/or not using the column and perform an isopropanol precipitation instead…

And/or make fresh stock of ampicillin (current stock is many years old, but has been frozen).

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RNA Isolation – Olympia oyster gonad tissue in paraffin histology blocks

UPDATE 20170712: The RNA I isolated below is from incorrect regions of tissue. I misunderstood exactly what this tissue was, and admittedly, jumped the gun. The tissue is actually collected from the visceral mass – which contains gonad (a small amount) and digestive gland (a large amount). The RNA isolated below will be stored in one of the Shellfish RNA boxes and I will isolate RNA from the correct regions indicated by Grace

Isolated RNA from Olympia oyster gonad previously preserved with the PAXgene Tissue Fixative and Stabilizer and then embedded in paraffin blocks. See Laura’s notebook for full details on samples and preservation.

 

RNA was isolated from the following samples using the PAXgene Tissue RNA Kit (Qiagen). Gouged samples from the blocks weighing ~10mg from each of the tissues and processed according the protocol for isolating RNA from blocks of paraffin-embedded tissues.

Tissue identification is available in this GitHub Issue

NF-10-22
NF-10-23
NF-10-24
NF-10-26
NF-10-28
NF-10-30
SN-10-16
SN-10-17
SN-10-20
SN-10-25
SN-10-26
SN-10-31

IMPORTANT:

  • Prior to beginning, I prepared an aliquot of Buffer TR1 by adding 40μL of β-mercaptoethanol (β-ME) to 4000μL of Buffer TR1).
  • Reconstituted DNase I with 550μL of RNase-free H2O. Aliquoted in 100μL volumes and stored @ -20C in the “-20C Kit Components” box.

Isolated RNA according to the PAXgene Tissue RNA Kit protocol with the following alterations:

  • “Max speed” spins were performed at 20,000g.
  • Tissue disruption was performed by adding ~25-50 glass beads (425 – 600μm diameter) with the Disruptor Genie @ 45C for 15mins (in the Friedman Lab).
  • Samples were eluted with 27μL of Buffer TR4 x 2, incubated @ 65C for 5mins, immediately placed on ice and quantified on the Roberts Lab Qubit 3.0 with the RNA High Sensitivity Assay (ThermoFisher Scientific) using 5μL of each sample.

Results:

Concentrations (Google Sheet): 20170710_RNA_qubit_oly_histo_blocks

Well, the good news is that there’s RNA from all the samples and it seems to be in relatively high concentrations!

The bad news is that the concentrations for 10 of the 12 samples were too high and outside the range of the Qubit RNA HS Assay! Since we don’t have the broad range RNA assay, I can’t properly quantify the remaining samples. However, these samples are being sent to Katherine Silliman at some point, so I’ll leave it up to her to quantify the samples. I’m also guessing that she’ll run them on a Bioanalyzer to assess their integrity prior to beginning library construction, so that will also yield concentrations for the samples.

Samples were stored at -80C temporarily.

Samples will be sent to Katherine Silliman for high-throughput library construction and sequencing once I hear back from her regarding her availability to receive the samples.

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DNA Quantification – Acropora cervicornis (Staghorn coral) DNA from Javier Casariego (FIU)

I quantified the three samples (listed below) that I SpeedVac’d yesterday using the the Roberts Lab Qubit 3.0.

  • 2h Block 1
  • 2h Block 8
  • D35 Block 8

Quantification was performed using the dsDNA Broad Range Kit.

Used 1uL of each sample.

Results:

One sample (2h Block 1) is still slightly too dilute in order to use the recommended total amount of DNA for the methylation assay (100ng), but still falls well within the recommended range for the assay. Will proceed with the methylation assay for all samples.

Values were added to the spreadsheet provided by Javier (Google Sheet): A.cervicornis_DNA_Extractions(May_2017).xlsx

 

Qubit output file (Google Sheet): 20170511_qubit_A_cervicornis_DNA

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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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