Received Caligus tape DNA – two samples:
- Female 1 .
- Female 2 .
Stored in slots H4 and H5 in “Sam’s gDNA Box #2″ in the FTR 213 -20oC freezer.
Google Sheet: Sam’s gDNA Box #2
Received genomic DNA from oysters infected with OsHV-1 variants.
Received the following samples with Australian (Aus) and French (M) variants from Carolyn (stored in my -20C Box #2):
Received the following samples with California variant from Natalie Rivlin (University of Maryland) (stored in -20C in FSH 240):
Used the MethylFlash Methylated DNA Quantification Kit (Colorimetric) from Epigentek to quantify methylation in these coral DNA samples.
All samples were run in duplicate <em>except</em> 2h Block 1 due to insufficient DNA.
The following samples were used in a 1:10 dilution (2uL DNA : 18uL NanoPure H2O), due to their relatively high concentrations, to ensure accurate pipetting:
All samples were diluted to a final concentration of 9.645ng/uL (154.24ng total; 17.6uL) in NanoPure water, which is equal to 77.12ng of DNA per assay replicate. These numbers were chosen based off of the sample with the lowest concentration.
The following samples were used in their entirety:
Calculations were added to the spreadsheet provided by Javier (Google Sheet): A.cervicornis_DNA_Extractions(May_2017).xlsx
The spreadsheet became overly complicated because I initially forgot to account for the need to run each sample in duplicate.
The kit reagent dilutions were as follows:
All diluted solutions were stored on ice for duration of procedure.
The remaining Diluted ME1 solution was stored at 4C (FTR 209), and is stable for 6 months, per the manufacturer’s instructions.
See the Results section below for plate layouts.
Plates were read at 450nm on the Seeb Lab Victor 1420 Plate Reader (Perkin Elmer) and the amount of DNA methylation was determined.
Individual sample methylation quantification (Google Sheet): A.cervicornis_DNA_Extractions(May_2017).xlsx
Plate Reader Output File Plate #1 (Google Sheet): 20170511_coral_DNA_methylation_plate01.xls
Plate Reader Output File Plate #2 (Google Sheet): 20170511_coral_DNA_methylation_plate02.xls
I’m not familiar with the experimental design, so I’m not going to spend time handling any of the in-depth analysis at this point in time. However, here’s the background on how methylation quantification and percent methylation were determined.
Mean absorbance (450nm) was determined for all samples and standard curve samples. It’s important to note that the standard deviation between replicates was not evaluated and there appears to be consistent variability between samples, but I’m not certain how much variation is “acceptable” with and assay of this nature.
The mean absorbance of the standard curve samples were plotted against their corresponding DNA amounts and a linear trendline was fitted to the points.
Per the manufacturer’s recommendations, the four points (including the zero point) that yielded the best linear fit (i.e. best R^2 value) were used and the slope of best fit line for those four points was determined.
This slope was then utilized in the equation provided by the manufacturer (see pg. 8 of the MethylFlash Kit manual).
Received 62 coral (Acropora cervicornis) DNA samples from Javier Casariego at FIU.
Spreadsheet of samples and NanoDrop concentrations provided by Javier (converted to Google Sheet): A.cervicornis_DNA_Extractions(May_2017).xlsx
Samples were temporarily stored at 4c (in FTR 213) until I can perform global methylation assessment on them tomorrow.
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.
Ran the coral DNA I quantified on 20160630 through the MethylFlash Methylated DNA Quantification Kit [Colorimetric] (Epigentek) kit to quantify global methylation.
Used 100ng of DNA per 8uL per replicate (x2 replicates = total 200ng in 16uL). Calcs are here (Google Sheet): 20160705_coral_DNA_methylation_calcs
Manufacturer’s protocol was followed.
Dilutions of kit reagents:
ME5 (1:1000) 2.6uL ME5 + 2597.4uL diluted ME1
ME6 (1:2000) 1.3uL ME6 + 2598.7uL diluted ME1
ME7 (1:5000) 0.52uL ME7 + 2599.48uL diluted ME1
Samples were quantified on the Seeb’s plate reader @ 450nm (Wallac 1420 Victor 2 [Perkin Elmer])
Google Sheet: 20160707_coral_DNA_methylflash
|H1_5||nitrogen & phosphorous||0.9663585942|
|H1_8||nitrogen & phosphorous||0.4244913398|
|H24_5||nitrogen & phosphorous||0.1495527697|
|H24_8||nitrogen & phosphorous||0.2213437801|
|H5_5||nitrogen & phosphorous||-0.07790933048|
|H5_8||nitrogen & phosphorous||0.5949647121|
Overall, it’s difficult to really interpret these results. I believe the data is a time course (e.g. H5 = hour 5, H24 = hour 24). Additionally, looking at treatments, there appear to be replicates, but it’s not clear what type of replicates they are (i.e. technical or biological). Generally, it seems like the control samples have lower quantities of methylated DNA than the treated samples. However, this doesn’t hold true for all three of the groups.
And, not that it really matters, but I don’t even know what species this is…
In any case, this was an attempt to gather some preliminary data for a grant that Steven is attempting to put together, so the original experiment and the subsequent data aren’t as robust as one would expect for a full-blown research project.
Quantified the DNA we received from Jose on 20160615 using the Qubit 3.0 Flouorometer (ThermoFisher) with the dsDNA Broad Range (BR) Kit according to the manufacturer’s protocol. Used 1μL of each sample.
Results are here (Google Sheet): Coral_DNA_QubitData_2016-06-30_08-45-56.xls
Here is a table of sample concentrations:
Will proceed with DNA methylation assessment.
Steven received these coral DNA samples today. Here’s his post on Google Plus (stored @ 4C in FTR 213):
Here’s the email from Jose describing the samples:
“Dear Steven, the coral DNA samples were sent today by my student Javier (cc’ed here) to your lab. Here’s an excel attached with info for the samples including concentration and treatment of the coral from which they were extracted (N, nitrogen; NP, nitrogen+phosphorous; C, control).
Please let us know when you get these in the lab so we know all is fine!
Here’s the spreadsheet he sent (renamed for easier identification later on – original file sent was title DNA Qbit readings), uploaded to Google Drive:
Samples were stored @ -20C in FTR 209.
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:
Tell sqlite that the field separator will be commas (i.e. CSV file):
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:
Set output display to include column headers:
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|
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.