Samples Received – White Abalone DNA from CDFW Shellfish Health Lab

Received white abalone (Haliotis sorenseni) DNA extracted from digestive gland, post-esophagus, and feces from Jim Moore and Blythe Marshman at the California Dept. of Fish & Wildlife.

These are intended for qPCR to assess presence of the RLOv.

Samples were stored in the big -20C in FSH 240.

 

DNase Treatment – Abalone Water Filters for RLO Viability

The RNA I isolated earlier today was subjected to DNase treatment using the Turbo DNA-free Kit (Invitrogen), following the manufacturer’s standard protocol.

After DNase inactivation treatment, the RNA was transferred (recovered ~19uL from each samples)  to a clear, low-profile PCR plate.

The plate layout is here (Google Sheet): 20170309_RLO_viability_DNased_RNA_plate_layout

The samples will be subjected to qPCR to assess the presence/absence of residual gDNA. The plate of DNased RNA was stored @ -80C in the original box that the water filters were stored in.

An overview of the experiment and the various treatments are viewable in the “Viability Trial 3″ tab of Lisa’s spreadsheet (Google Sheet): RLO Viability & ID50

RNA Isolation – Abalone Water Filters for RLO Viability

Water filters stored at -80C in ~1mL of RNAzol RT were provided by Lisa. This is part of an experiment (and Capstone project) to assess RLO viability outside of the host.

The samples were thawed and briefly homogenized (as best I could) with a disposable plastic pestle. The samples were then processed according to the manufacturer’s protocol for total RNA isolation. Samples were resuspended in 25μL of nuclease-free water (Promega).

Immediately proceeded to DNase treatment.

The experimental samples and the various treatments are viewable in the “Viability Trial 3″ tab of Lisa’s spreadsheet (Google Sheet): RLO Viability & ID50

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.

 

 

 

 

 

 

 

 

 

Curriculum Testing – Determination of Most Useful Concentration of Sodium Carbonate Solution

After evaluating whether or not dry ice would be effective to trigger a noticeable change in pH in a solution, I determined which concentration(s) of sodium carbonate (Na2CO3) would be most useful for demonstration and usage within the curriculum. Previously, I used a 1M Na2CO3 solution a the universal pH indicator showed no change in color. What I want is a color change, but one that takes place at a noticeably slower rate than the other solutions that are demonstrated/tested; this will show how sodium carbonate acts as a buffer to CO2-acidification.

Additionally, I tested the difference in rate of pH change between Instant Ocean and sodium chloride (NaCl). The reason for testing this is to use this as a demonstration that salt water (i.e. sea water, ocean water) isn’t just made up of salt. It’s likely that many students simply think of the ocean as salt water and have not considered that the makeup of sea water is much more complex.

Finally, I performed these tests in larger volumes than I did previously to verify that the larger volumes will slow the rate of pH change, thus increasing the time it takes for the universal pH indicator to change color, making it easier to see/monitor/time.

Instant Ocean mix (per mfg’s recs): 0.036g/mL (36g/L)

For the NaCl solution, I used the equivalent weight (36g) that was used to make up the Instant Ocean solution.

 

Results:

  • Use of 0.001M Na2CO3 is passable, but due to the fact that it’s a diprotic base, the pH indicator didn’t progress lower than ~pH 6.0 in my limited tests. Adding additional dry ice (or using an even more dilute solution) are options to drive the pH lower.
  • The comparison between salt water and Instant Ocean will work well as a demonstration to introduce the concept that sea water is more complex than just being salty.
  • Using 1L volumes works well to slow the color changes of the universal pH indicator to improve the ability of the students to observe and measure the rate of color change.

The table below summarizes what I tested.

SOLUTION VOLUME (mL) DRY ICE (g) TIME OBSERVATIONS
0.1M Na2CO3 1000 3.0 No color change. Dry ice gone.
0.01M Na2CO3 1000 3.3 No color change. Dry ice gone.
0.001M Na2CO3 1000 3.3 ~20s Dry ice gone, but final color indicated a pH ~6.0.
Instant Ocean 1000 3.3 3m Initial color change noticeable within 10s; full color change after ~3m
NaCl 1000 3.0 instant Immediate, complete color change.
Tap H2O 1000 3.3 3m pH started @ ~7.5. Full color change took place.

Curriculum Testing – Viability of Using Dry Ice to Alter pH

Ran some basic tests to get an idea of how well (or poorly) the use of dry ice and universal indicator would be for this lesson.

Instant Ocean mix (per mfg’s recs): 0.036g/mL

Universal Indicator (per mfg’s recs): 15μL/mL

Played around a bit with different solution volumes, different dry ice amounts, and different Universal Indicator amounts.

Indicator Vol (mL) Solution Solution Vol (mL) Dry Ice (g) Time to Color Change (m) Notes
3 Tap H2O 200 1.5 <0.5
3 Tap H2O 200 0.5 >5 Doesn’t trigger full color change and not much bubbling (not very exciting)
5 Tap H2O 1000 12 <1
3 Instant Ocean 200 1.5 <0.5 Begins at higher pH than just tap water. Full color change is slower than just tap water, but still too quick for timing.
2 1M Na2CO3 200 5 >5 No color change and dry ice fully sublimated.
2 1M Tris Base 200 5 >5 No color change and dry ice fully sublimated.
2 Tap H2O + 20 drops 1M NaOH 200 5 2.75 ~Same color as Na2CO3 and Tris Base solutions to begin. Dry ice gone after ~5m and final pH color is ~6.0.

 

Summary

  • Universal Indicator amount doesn’t have an effect. It’s solely needed for ease-of-viewing color changes. Use whatever volume is desired to facilitate easy observations of color changes.
  • Larger solution volumes should be used in order to slow the rate of pH change, so that it’s easier to see differences in rates of change between different solutions.
  • 1M solutions of Na2CO3 and Tris Base have too much buffering capacity and will not exhibit a decrease in pH (i.e. color change) from simply using dry ice. May want to try out different dilutions.
  • Use of water + NaOH to match starting color of Na2CO3 and/or Tris Base is a good way to illustrate differences in buffering capacity to students.
  • Overall, dry ice will work as a tool to demonstrate effect(s) of CO2 on pH of solutions!

Some pictures (to add some zest to this entry):

 

 

 

Hard Drive Replacement – Microscrope Computer (Dell Optiplex GX620)

Dan noticed that the computer wouldn’t boot, so I looked into it a bit. When attempting to boot, the hard drive (HDD) was making a clicking noise; this is never a good sign.

I replaced the HDD with a clone of the existing (now dead) HDD that I had created back on 20150422 and everything is mostly back to normal.

What hasn’t returned to normal is the usage of Dropbox. Sometime this summer, Dropbox stopped supporting Windows XP and no longer allows usage of the Dropbox app on Windows XP computers. For the time being, this means that all files saved on this computer should be uploaded to Dropbox via a web browser.

Saving files to the Dropbox folder that still exists on this computer will NOT sync! That means they will NOT be backed up.

To resolve this issue, we would need to upgrade to Windows 7. Once I obtain a new backup HDD to create a new clone, I’ll attempt to upgrade this computer to Windows 7. The main reservation I have about this is that the two key pieces of software installed on this computer (Nikon Elements and SPOT) are extremely old and may not function on a newer Windows version. But, I guess we won’t know until we try!

Below are images of the steps I took to replace the dead HDD:

 

 

 

 

 

 

 

qPCR – RLOv DNA helicase and XenoCal prophage on Ab Endo Water Filters

Stan Langevin was interested in seeing if the RLOv (phage) and/or the prophage portal genes were detectable in water samples from Lisa’s Ab Endo project.

Ran qPCR on the following samples that Lisa selected:

DNA from water filters collected in 2010. DNA isolated 20120111:

  • CP 0M A
  • CP 0M B
  • MA 0M A
  • MA 0M B
  • PSN 0M A
  • PSN 0M B
  • RM A
  • RM B

DNA from water filters collected in 2011. DNA isolated 20140822:

  • AM Drain 2B
  • PCI SRI PC 1B

RLOv_DNA_helicase master mix calcs are here (Google Sheet): 20161213 – qPCR RLOv DNA Helicase

XenoCal prophage master mix calcs are here (Google Sheet): 20161213 – qPCR XenoCal phage portal

RLOv_DNA_helicase standard curve from 20151224.

All samples were run in duplicate. Plate layout, cycling params, etc. can be seen in the qPCR Report below.

Results:

RLOv_DNA_helicase
qPCR Report (PDF): Sam_2016-12-13 14-52-05_CC009827_RLOv_helicase.pdf
qPCR Data File (CFX): Sam_2016-12-13 14-52-05_CC009827_RLOv_helicase.pcrd

 

XenoCal prophage
qPCR Report (PDF): Sam_2016-12-13 14-52-05_CC009827_XCprophage.pdf
qPCR Data File (CFX): Sam_2016-12-13 14-52-05_CC009827_XCprophage.pcrd

 

  • RLOv DNA helicase amplified in all samples EXCEPT the two samples from 2011. These two samples were negative for the RLO (see Ab Endo sheet “water 2011″).
  •  XC prophage amplfied inconsistently (i.e. replicates did not match/amplify) in only three samples. Additionally, the melt curve of one of those samples differs from the other two. Based on the inconsistencies in technical reps, I should probably repeat this, but technical reps across all of the RLOv DNA helicase samples are very tight, suggesting that my technique was fine (it would be odd if my technique faltered only on ALL of the XC prophage samples)…

 

RLOv DNA HELICASE

 


 

XENOCAL PROPHAGE

 

qPCR – LCM DNA

Ran three primer sets on laser capture microscopy (LCM) DNA samples from 2005 and 2007. Ran the following primer sets:

  • WSN1 (detects RLO)
  • RLOv_helicase (detects RLO phage)
  • XenoCal_prophage

The DNA samples were provided to me by Lisa. I’m not entirely sure of their history:

 

Master mix calcs (Google Sheets):

All samples were run in duplicate. Plate layout, cycling params, etc. are in the qPCR Reports (see Results below).

Standard curves:

Baseline threshold was manually set to 580 for the WSN1 samples, as previously determined by Lisa for this assay.

Baseline threshold was manually set to 580.5 for the RLOv DNA helicase samples, as previously determined by me on 20160128.

 

Results:

WSN1:

 

RLOv DNA helicase:

 

XenoCal prophage:

 

Summary table of all three genes in each sample. Unfortunately, I don’t fully understand the sample name nomenclature, so I can’t really come to any conclusions about the data. Will pass along to Carolyn, Lisa, and Stan.

It’s also important to note that, due to low sample volume, I did not quantify these samples. This is important because any samples listed below that are negative for all three genes can not be conclusively declared “negative”, since we can’t rule out the possibility that they simply lack any DNA.

Presumably they were quantified after their initial extraction?

SAMPLE WSN1 RLOv DNA HELICASE XC PROPHAGE
LCM New RLO 09 + + +
LCM ST RLO 09 - - -
LCM New 08:30-5 B + + +
LCM New 08:30-5 - - -
LCM ST 08:30-3 - - -
LCM WS RLO + - +

 

STANDARD, AMPLIFICATION, & MELT CURVES

 

WSN1

 

 


 

 

RLOv_DNA_helicase

 


 

 

XenoCal prophage

qPCR – Water Filter cDNA for RLO Viability Assessment

Ran qPCRs on the cDNA I made earlier today to determine if there’s any detectable RNA in any of these water filter samples.

Master mix calcs (Google Sheet): 20161208- qPCR WSN1

All samples were run in duplicate. Plate layout, cycling params, etc. are in the qPCR Report (see Results below).

Standard curve was the p18RK7 curve made on 20161128.

Baseline threshold was manually set to 580, as previously determined by Lisa for this assay.

Results:
qPCR Report (PDF): Sam_2016-12-08 09-14-38_CC009827_cDNA_WSN1.pdf
qPCR File (CFX96): Sam_2016-12-08 09-14-38_CC009827_cDNA_WSN1.pcrd

Original qPCR File (CFX96): Sam_2016-12-08 09-14-38_CC009827.pcrd

Standard curve looks good.

The following cDNA samples had detectable amplification:

  • T1A
  • T1B
  • T3A
  • T3B

I believe that the labelling scheme represents T1 = Day 1 in water, T3 = Day 3 in water.

These results suggest that the RLO is viable outside of the abalone host for at least three days, but not >= 7 days, although the values are below the theoretical qPCR limit of detection. These results will likely be used to help Lisa with experimental design for a more involved assessment of RLO viability in the water column.

I’ve added the data to Lisa’s spreadsheet (Google Sheet: RLO viability) in the “Expt 1″ worksheet.

Update after talking to Lisa: The water was shipped from a California abalone farm O/N, so T0 = 24hr water. The Control water samples were sea water from our basement facility, not from California.

The fact that there is no amplification at T0 is a bit surprising and possibly suggests that RLO viability outside of the host is on the magnitude of hours, not days…