Tag Archives: tissue

Sample Prep – Pinto Abalone Tissue/RNA for Collabs at UC-Irvine

We need to send half of each sample that we have from Sean Bennett’s Capstone project to Alyssa Braciszewski at UC-Irvine.

This is quite the project! There are ~75 samples, and about half of those are tissues (presumably digestive gland) stored in RNAzol RT. The remainder are RNA that has already been isolated. Additionally, tube labels are not always clear and there are duplicates. All of these factors led to this taking an entire day in order to decipher and process all the samples.

I selected samples from only those that I was confident in their identity.

I aliquoted 25μL of each RNA for shipment to Alyssa.

Tissue samples were thawed and tissue was cut in half using razor blades.

Planning to send samples on Monday.

Lisa has already assembled a master spreadsheet to try to keep track of all the samples and what they are (Google Sheet): Pinto Transcriptome

Here’s the list of samples I’ll be sending to Alyssa (Google Sheet): 20170222_pinto_abalone_samples

Here are some images to detail some of the issues I had to deal with in sample ID/selection.

 

 

 

 

 

 

 

 

 

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Another day, another species

Today we sampled Jesse, the Olympia oyster selected for genome sequencing. Not as big of a specimen as yesterday, but made up for it with video.

The oyster was collected in Fidalgo Bay, weighing in at 58.99 grams and 70 mm long.

The adductor muscle was selected for genome sequencing, thus quickly rinsed in 10% bleach.

Other tissues were sampled for RNA-seq

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Samples were placed in two boxes, for redundancy.
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Big day, big clam

Today we sampled the geoduck (Panopea generosa) for genome sequencing. Here is how things went down.

It was an early morning for the clam, peaking out to see a glorious sunrise on the porch.
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From there it was off to the lab.

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After cleaning the surfaces, Brent sampled tissue.

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We started out started out targetting the foot and adductor muscles. These tissues were steriley removed and then rinsed in 1% bleach, followed by Nanopure water. This tissue will be used for genome sequencing as we predict least amount of associated taxa.

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Remaining tissues were taken, primarily for RNA-seq and divided into two boxes.

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Tubes were labeled on cap with tissue type.
Here is what Box 1 looks like.

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Box 2 looks the same however it does not have a heart or style sample.

The only surpise was in sampling, labial palps were identified after we had already sampled a pair.

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qPCR – Withering Syndrome qPCR Assay Validation: Reproducibility (CFX 96)

Ran qPCR for the reproducibility aspect of the WSN qPCR Assay Validation. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the Results (see below).

Standard curve was the p16RK7 NcoI-linearized curve made on 20120730.

Baseline threshold was set to 400 and cycles to analyze was set to 41.

Samples used for “low”, “medium” and “high” copy numbers for each sample type are below, with expected fold copy number (based off of previous qPCRs):

Feces

Low: R4E 4/17/09 – 10^0

Med: R3E 7/23/09 – 10^3

High: R4E 7/23/09 – 10^4

Tissue

Low: 09:16-18 – 10^1

Med: 09:16-22 – 10^2

High: 09:20-11 – 10^5

Water

Low: 494:11-11 – 10^0

Med: 494-11-12 – 10^2

High: TAF SD A2 – 10^3

Results:

qPCR Data File (CFX96): Sam_2012-10-22 16-16-19_CC009827.pcrd

qPCR Report (PDF): Sam_2012-10-22 16-16-19_CC009827.pdf

Everything looked good except for the Low Feces sample which didn’t produce any amplification. Will identify another sample to use for the Low Feces sample.

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