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.