Tag Archives: pics

qPCR – Black Abalone DNA with Varying Levels of RLO/RLOv

Carolyn & Stan Langevin have agreed that the DNA helicase qPCR should be tested on 10 black abalone DNA extractions that fall into multiple levels of the Friedman Lab’s withering syndrome histology scoring.

Earlier today I identified samples at three different histology scoring levels of RLO: 0, 1, & 2.

Here’s the list of samples that will be qPCR’d. There were only eight samples that had histology scores of 2 in both PE and Dg.

RLO/RLOv 0 RLO/RLOv 1 RLO/RLOv 2
06:5-18 06:5-35 06:5-31
06:5-30 06:6-32 06:5-32B
06:50-04 06:6-39 06:6-46
06:50-05 06:6-42 06:6-49
07:12-01 06:6-44 08:3-05
07:12-02 06:6-52 08:3-07
07:12-03 06:6-54 08:3-15
07:12-04 06:50-08 08:3-16
07:12-07 06:50-10
07:12-09 07:12-18

 

Ran qPCR using the RLOv DNA helicase standard curve from 20151106.

All samples were run in duplicate on the CFX96 (BioRad).

Master mix calcs are here: 20151120 – qPCR RLOv Black Abs

Plate layout, cycling params, etc. can be seen in the qPCR Report (see Results).

Results:
qPCR Report (PDF): Sam_2015-11-20 15-00-27_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-11-20 15-00-27_CC009827.pcrd

Quick summary of the results:

  • 50% of the RLO/RLOv 0 score samples are positive for RLOv DNA helicase. Will talk to Carolyn to see if she has withering syndrome qPCR data for these samples to compare RLOv-positive samples with WSN-positive samples. If not, will run withering syndrome qPCR.
  • All RLO/RLOv 1 & 2 scored samples are positive for RLOv DNA helicase
  • All RLO/RLOv 2 scored samples come up before the standar curve; these should be diluted and re-run.
  • Standard curve isn’t perfect (the 3 copy sample is throwing it off).

 

STANDARD CURVE AMP & SCATTER PLOTS

 

 

RLO/RLOv 0 AMP PLOTS

 

 

RLO/RLOv 1 AMP PLOTS

 

 

RLO/RLOv 2 AMP PLOTS

Share

Sample ID – Black Abalone DNA for RLOv qPCRs

Carolyn & Stan Langevin have agreed that the DNA helicase qPCR should be tested on 10 black abalone DNA extractions that fall into multiple levels of the Friedman Lab’s withering syndrome histology scoring.

Downloaded the (Google Sheet) Black Abalone: Expt 1 – WS & Phage as a CSV file. After downloading, I renamed the file (Black_Abalone.csv) to facilitate easier usage in the following steps.

Created a sqlite database using GitBash for Windows:
Change to directory where file is located:

$cd Downloads

Start sqlite:

$sqlite3

Tell sqlite that the field separator will be commas (i.e. CSV file):

sqlite>.separator ","

Import the CSV file and provide a name for the resulting database:

sqlite>.import Black_Abalone.csv BlackAbs

Set output display mode to column for easier reading:

sqlite>.mode column

Set output display to include column headers:

sqlite>.headers on

 

To select all the samples that have scores of 0 in both PE and DG RLO fields (screen cap does not show entire output list):

 

To select all the samples that have scores of 1 in both PE and DG RLO fields:

 

To select all the samples that have scores of 2 in both PE and DG RLO fields:

 

Here are the full set of results in a table

RLO/RLOv 0 RLO/RLOv 1 RLO/RLOv 2
06:5-03 06:5-35A 06:5-31
06:5-04 06:50-08 06:5-32B
06:5-08 06:50-10 06:6-46
06:5-09 06:6-32 06:6-49
06:5-10 06:6-39 08:3-05
06:5-11 06:6-42 08:3-07
06:5-14 06:6-44 08:3-15
06:5-16 06:6-52 08:3-16
06:5-18 06:6-54
06:5-20 07:12-18
06:5-21 08:3-08
06:5-22 08:3-10
06:5-24
06:5-30
06:50-04
06:50-05
06:50-11
06:50-12
06:50-13
06:50-15
06:50-16
06:6-01
06:6-02
06:6-03
06:6-05
06:6-08
06:6-11
06:6-12
06:6-13
06:6-15
06:6-16
06:6-17
06:6-18
06:6-20
06:6-21
06:6-22
06:6-23
06:6-24
06:6-25
06:6-26
06:6-27
06:6-28
07:12-01
07:12-02
07:12-03
07:12-04
07:12-05
07:12-06
07:12-07
07:12-09
07:12-10
07:12-13
07:12-19
08:3-01
08:3-02
08:3-03
08:3-04
08:3-13
08:4-01
08:4-02
08:4-03
08:4-04
08:4-05
08:4-06
08:4-07
08:4-08
08:4-09
08:4-10
08:4-11
08:4-12
08:4-13
08:4-14
08:4-15
08:4-16
08:4-17
08:4-18
08:4-19
08:4-20
08:4-21
08:4-22
08:4-23
08:4-24
08:4-25
08:5-06

Will select just 10 of those in the RLO/RLOv 0 column for use in qPCR.

I was able to track down the boxes where are these DNAs were stored (see images below).

Boxes that were not labeled with accession numbers of the samples contained therein are now labeled.

Boxes that contained samples that belonged in other boxers were transferred to the appropriate box.

All boxes were located, and returned, to the big -20C in 240 on Lisa’s shelf.

Share

Data Management – Black Abalone Histology Scores

As part of the qPCR validation for the withering syndrome phage (RLOv) project, I needed to identify (and, eventually locate) samples that are infected with varying levels of RLOv. This is probably the most time consuming aspect of the project.

I found the histology scoring sheets and added them to an existing Google Sheet that Lisa had partially completed a few years ago: Black Abalone: Expt 1 – WS & Phage

To save time, I only entered the scores into the spreadsheet and did not enter any extra info (like Sex or Coccidia).

Having this data in a single, digital format will allow me to sort the data, to quickly & easily select the appropriate samples with varying levels of RLOv (categorized as “New” on the sheet).

Here are links to pics of the histology scoring sheets for reference:

Next up will be to actually track down the physical samples. This will be a bit of a daunting task…

Share

PCR – RLOv Clones

Colony PCRs were performed on each of the transformations from 20151015 (RLOv_ DNA_helicase, RLOv_head_to_tail, RLOv_membrane_gene_1, RLOv_membrane_gene_2, RLOv_tail_to_fiber) to confirm successful ligations in plasmid pCR2.1 using the M13F/R vector primers.

Colonies were picked form the transformation plates with pipette tips, re-streaked on a secondary, gridded, numbered LBAmp100+x-gal plate and then used to inoculate the respective PCR reactions.

Six white colonies (positive clones) and a single blue colony (negative clone) were selected from each transformation.

Master mix calcs are here (Google Sheet): 20151019 – Colony PCRs RLOv

Restreaked plates were incubated @ 37C O/N and then stored @ 4C (Parafilmed).

30μL of each reaction was run on a 1% agarose 1x Low TAE gel, stained w/EtBr.

Results:

 

All the PCRs look good. All white colonies selected contain a PCR product of appropriate size (i.e. larger than the blue colonies; negative [-C] control). Will select clones #1 from each to grow up for plasmid prep.

Share

Abalone Sampling – Post-esophagus & Digestive Gland

Helped Ava sample 162 red abalone (Haliotis rufescens).

Accession numbers 15:11-1 through 15:11-162

  • Animals were weighed.
  • Took one post-esophagus sample and one digestive gland sample for histology, placed in histology cassette; three animals per cassette.
  • Took one post-esophagus sample in 95% ethanol for qPCR
  • Shells were labeled and retained for post-sampling weighing/measurements.

Share

Abalone Sampling – Post-esophagus & Digestive Gland

Helped Ava sample 74 red abalone (Haliotis rufescens).

Accession numbers 15:10-1 through 15:10-74

  • Animals were weighed.

  • Took one post-esophagus sample and one digestive gland sample for histology, placed in histology cassette; three animals per cassette.

  • Took one post-esophagus sample in 95% ethanol for qPCR

  • Shells were labeled and retained for post-sampling weighing/measurements.

Share

RNA Isolation – Jake’s O. lurida Ctenidia 1hr Heat Stress from 20150422

Isolated RNA from Jake’s Olympia oyster ctenidia, 1hr heat shock, collected on 20150422. Samples had been homogenized and stored @ -80C.

The following sample tubes (heat-shocked oyster ctenidia) were removed from -80C and thawed at RT:

  • 42215 HT1 1
  • 42215 HT1 2
  • 42215 HT1 3
  • 42215 HT1 4
  • 42215 HT1 5
  • 42215 HT1 6
  • 42215 HT1 7
  • 42215 HT1 8
  • 42215 NT1 1
  • 42215 NT1 1
  • 42215 NT1 2
  • 42215 NT1 3
  • 42215 NT1 4
  • 42215 NT1 5
  • 42215 NT1 6
  • 42215 NT1 7
  • 42215 NT1 8
  • 42215 ST1 1
  • 42215 ST1 2
  • 42215 ST1 3
  • 42215 ST1 4
  • 42215 ST1 5
  • 42215 ST1 6
  • 42215 ST1 7
  • 42215 ST1 8

NOTE: Samples NT1 1 and NT1 2 only had 700μL of RNAzol RT in them. Added additional 300μL of RNAzol RT to each.

NOTE: 0.1% DEPC-H2O used throughout this procedure was prepared on 7/15/2010 by me.

According to Jake’s notebook entry, the samples should have been previously homogenized in RNAzol RT. However, none of the samples showed evidence of being homogenized:

 

In theory, if these samples were snap frozen on liquid nitrogen after being placed in the RNAzol RT, there should be almost no impact on the RNA.

 

Procedure:

Samples were homogenized with disposable pestle in their respective tubes and vortexed.

Added 400μL of 0.1% DEPC-H2O to each sample and vortexed 15s.

Incubated samples 15mins at RT.

Centrifuged tubes 15mins at RT @ 16,000g.

750μL of the supe was transferred to a clean tube, added equal volume of isopropanol (750μL), mix by inversion (20 times), and incubated at RT for 15mins.

Centrifuged 12,000g for 10mins.

Discarded supe.

Washed pellets with 500μL of 75% EtOH (made with 0.1% DEPC-H2O) and centrifuged 4,000g for 3mins at RT. Repeated one time.

Removed EtOH and resuspended in  100μL of 0.1% DEPC-H2O. Most samples required vortexing to dissolve pellet.

Sample tubes were transferred to ice, quantified on the Roberts Lab NanoDrop1000, and stored @ -80C in their original box, pictured:

 

Results:

 

Google Spreadsheet with absorbance data: 20150506_Jake_Oly_1h_HS_RNA_ODs

Overall, the samples have excellent yields. The exceptions being the two samples that had less than 1mL of RNAzol RT in them to start (their yields are actually fine, but relative to all the other samples, they aren’t great). Should I have left them that way instead of adding additional RNAzol RT? Was there something wrong with these samples in the first place and that’s why they didn’t have a full 1mL of RNAzol RT in the tube already?

The 260/280 ratios are pretty good for most of the samples (>1.8), however I’d prefer to see RNA with 260/280 ratios >1.9.

The 260/230 ratios are amazing! The best I’ve seen coming straight out of an RNA isolation in a long time.

Eventually (once I’ve isolated RNA from the control set that corresponds to these heat shock samples), I’ll check for gDNA carryover and then, probably, DNase the RNA.

Share