Set up qPCR with EF1 primers and IL17 Internal primers. Plate layout/setup is here. Note: gDNA sample used as a “positive” control will NOT amplify with the EF1 primers.
Results: Processed with PCR Miner. Normalized to EF1. Standard Error bars. Here is spreadsheet with workup.
IL17 Internal:
No significant differences between any treatments.
This is being done to check whether or not there is gDNA contamination in these cDNA and DNased RNA. Will use H.crach_h-1fg_intron primers. gDNA 07:12-15 was used as a positive control, based on results from yesterday’s qPCR. qPCR plate layout/set up is here. Anneal temp 50C.
Results: Everything came up negative, including the positive control! Also, the machine experienced an error at ~cycle 39, so no melting curve info. See below.
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). 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) and a 0.1s (Excel file) read. Both reads have nearly identical values.
