Tag Archives: MethylFlash Methylated DNA Quantification Kit [Colorimetric]

DNA Methylation Quantification – Acropora cervicornis (Staghorn coral) DNA from Javier Casariego (FIU)

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.

Results:

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.

  1. 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.

  2. The mean absorbance of the standard curve samples were plotted against their corresponding DNA amounts and a linear trendline was fitted to the points.

  3. 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.

  4. This slope was then utilized in the equation provided by the manufacturer (see pg. 8 of the MethylFlash Kit manual).

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DNA Methylation Quantification – Coral DNA from Jose M. Eirin-Lopez (Florida International University)

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])

Results:

Google Sheet: 20160707_coral_DNA_methylflash

sample treatment 5-mC(ng)
H1_1 nitrogen 0.8712248853
H1_10 nitrogen 0.6917168368
H1_12 control 0.2738478893
H1_5 nitrogen & phosphorous 0.9663585942
H1_6 control 0.6494783825
H1_8 nitrogen & phosphorous 0.4244913398
H24_1 nitrogen 0.372603297
H24_10 nitrogen 0.4237237786
H24_12 control 0.5350511937
H24_5 nitrogen & phosphorous 0.1495527697
H24_6 control 0.2291900804
H24_8 nitrogen & phosphorous 0.2213437801
H5_1 nitrogen -0.1233169902
H5_10 nitrogen 0.6997668774
H5_12 control 0.2307000493
H5_5 nitrogen & phosphorous -0.07790933048
H5_6 control 0.4562401662
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.

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