Used BB & DH samples #11-17 for procedure. Followed Epigentek’s protocol. My calcs for dilutions/solutions needed are here. All solutions were made fresh before using, except for Diluted GU1 which was made at the beginning of the procedure and stored on ice in a 50mL conical wrapped in aluminum foil. Used 100ng total (50ng/uL) of each sample gDNA. No standards for a standard curve based on speaking with Mac.
| WELL | SAMPLE | WELL | SAMPLE |
| A01 | BB11 | A02 | DH11 |
| B01 | BB12 | B02 | DH12 |
| C01 | BB13 | C02 | DH13 |
| D01 | BB14 | D02 | DH14 |
| E01 | BB15 | E02 | DH15 |
| F01 | BB16 | F02 | DH16 |
| G01 | BB17 | G02 | DH17 |
| H01 | Pos. Control | H02 | Blank |
Results: Above is the graph of the results. Although it’s only a small difference between the two sites, it is statistically significant. [The calcs for this graph can be found here (Excel file)(https://eagle.fish.washington.edu/Arabidopsis/Notebook%20Workup%20Files/20090519%20Gigas%20DNA%20methylation%20workup%20SJW.xls). It should be noted that this graph was generated using estimated values from the standard curve provided in the manufacturer’s protocol. This was done because 1) I did not run standards to generate my own curve and 2) calculating the “% methylation” not using the formula that utilizes the standard curve was giving ridiculously high values (e.g. 350%).
Here is the raw data generated by the plate reader for a [1s read (Excel file)(https://eagle.fish.washington.edu/Arabidopsis/Notebook%20Workup%20Files/20090519%20gDNA%20Methylation%201s%20SJW.xls) and a 0.1s (Excel file) read. Both reads have nearly identical values.