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:
- 72h Block 4
- D14 Block 1
- D14 Block 2
- D14 Block 3
- D14 Block 4
- D14 Block 5
- D14 Block 6
- D14 Block 8
- D14 Block 10
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:
- 2h Block 8
- D35 Block 8
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:
- Diluted ME1: 52mL of ME1 + 464mL of <em>distilled</em> water
- Diluted ME4: 10uL of ME4 + 10uL of TE Buffer (pH=8.0; made by me on 20130408).
- Standard curve: Prepped per instruction manual, with double volumes for two plates.
- Diluted ME5: 50uL/well x 152well = 7600uL; 7600uL/1000 = 7.6uL; 7.6uL ME5 + 7592.4uL Diluted ME1
- Diluted ME6: 50uL/well x 152well = 7600uL; 7600uL/2000 = 3.8uL; 3.8uL ME6 + 7596.2uL Diluted ME1
- Diluted ME7: 50uL/well x 152well = 7600uL; 7600uL/5000 = 1.52uL; 1.52uL ME7 + 7598.48uL Diluted ME1
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).