Data Aggregation – WS RLO and RLOv DNA Helicase qPCR and WS RLO Infection Intensities

Carolyn asked me to send her the data described above.

RLOv DNA helicase qPCR data were grabbed from the qPCR I ran on 20160106.

The qPCR data for withering syndrome RLO were culled from these three different spreadsheets:

 

The summary is below. I have emailed a copy of the spreadsheet to Carolyn.

Google Sheet: 20160404_Summary_RLO_RLOvDNAhelicase_qPCR_HistoIntensities

Sample ID – RLO+/RLOv- Samples for XenoCal Prophage Portal qPCRs

Earlier today I identified a bunch of samples that will be used to test out the XenoCal prophage portal primers to see if it’s present in the withering syndrome bacteria (RLO) or in the wither syndrome phage (RLOv). However, my search showed that we didn’t have sufficient samples that were RLO+/RLOv-.

I contacted Carolyn and she provided me with a spreadsheet summarizing the 2nd black abalone withering syndrome challenge. This data has samples that are known to be RLO+ (via qPCR – data is in spreadsheet) and should be RLOv- (will have to verify via qPCR before proceeding).

Google Sheet: MAIN data summary- black abalone 2nd study Nov  19 2012 for stats with question re trial 2 slides

It looks like the 08:13 accessions are all RLO+. I will track down these samples and verify they are RLOv- before proceeding with the XenoCal prophage portal qPCRs.

Sample ID- XenoCal Prophage Portal Tests

Now that the XenoCal prophage portal primers appear to be in working order, Carolyn wants me to test them out on 10 samples with the following status':

  • RLO-/RLOv-
  • RLO-/RLOv+
  • RLO+/RLOv-
  • RLO+/RLOv+

In order to quickly identify samples with these qualifications, I ran a SQL query on the following spreadsheet that contain qPCR data for both withering syndrome (RLO) and the phage (RLOv):

I saved the following worksheets from the above Google Sheet as CSV files:

  • water 2010
  • water 2011

These were imported to SQLite as I’ve previously done.

The two sheets were renamed for use in SQLite, respectively:

  • AbEndoWater2010
  • AbEndoWater2011

Here are the four queries I ran to obtain the four combinations of RLO/RLOv samples listed above

RLO-/RLOv-

sqlite> SELECT '2011_H2O', "DNA Tube Label", "Mean Cq", "RLOv_mean_Cq" FROM AbEndoWater2011 WHERE "Mean Cq"=0 AND "RLOv_mean_Cq"=0

 

RLO-/RLOv+

sqlite> SELECT '2011_H2O', "DNA Tube Label", "Mean Cq", "RLOv_mean_Cq" FROM AbEndoWater2011 WHERE "Mean Cq"=0 AND "RLOv_mean_Cq">0

 

RLO+/RLOv-

sqlite> SELECT '2011_H2O', "DNA Tube Label", "Mean Cq", "RLOv_mean_Cq" FROM AbEndoWater2011 WHERE "Mean Cq">0 AND "RLOv_mean_Cq"=0

 

RLO+/RLOv+

sqlite> SELECT '2011_H2O', "DNA Tube Label", "Mean Cq", "RLOv_mean_Cq" FROM AbEndoWater2011 WHERE "Mean Cq">0 AND "RLOv_mean_Cq">0

 

Results:

It looks like we do not currently have 10 samples that are RLO+/RLOv-. I will contact Carolyn to see if she happens to know of any samples that are RLO+, but do not contain (or, should not) any RLOv.

The full list of results can be seen in the Google Sheet below.

Google Sheet: 20160322_RLO_RLOv_pos_negs

qPCR – Check New Withering Syndrome p18RK7 Plasmid Standard Curve Dilution

This is a qPCR to test the fresh p18RK7 dilution I made earlier today, and verify it works well (i.e. linear spread, good R^2 value, same Cqs as previous curve dilution, etc.).

Master mix calcs (Google Sheet): 20150316 – qPCR WS p18RK7 Curve Check

I made three separate master mixes to check the curve three times.

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

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

Results:

qPCR Report (PDF): Sam_2016-03-16 17-04-05_CC009827.pdf
qPCR Data File (CFX): Sam_2016-03-16 17-04-05_CC009827.pcrd

Overall, the curve looks good and has very comparable Cq values at each dilution of the curve. Will use this for future withering syndrome qPCR assays and will send an aliquote of each dilution to Colleen Burge.

MASTER MIX #1


 

MASTER MIX #2

 


MASTER MIX #3

Sample Prep – Withering Syndrome p18RK7 Plasmid Standard Curve Dilution

We are virtually out of the withering syndrome qPCR standard curve plasmid dilutions and I need to send some to Colleen Burge at Univ. of Maryland Baltimore County. As such, I’ll need to make a fresh dilution series and test it out prior to sending to her.

Used NcoI-linearized p18RK7 abalone withering syndrome plasmid (from 20120726) to make a fresh dilution series.

Since it’s been nearly 4yrs since the plasmid was prepared, I decided to re-quantify the DNA using a Qubit3 (Life Technologies) in the Roberts Lab. Used the dsDNA BR Qubit reagents.

SAMPLE CONCENTRATION (ng/μL)
p18RK7 NcoI-linearized 29.0

The result is pretty close to what had been measured 4yrs ago (~26.1ng/μL – difference of  ~10%), so that’s good to see.

I prepared the dilution series using IDTE (pH=8.0; IDT) Buffer. The dilution worksheet below was set up by Nate Wight many, many, moons ago.

Dilution calcs (Google Sheet): 20160316 – WS p18RK7 plasmid dilution table