Finished EtOH precipitation of MeDIP gDNA. Samples were pelleted 16,000g, 4C, 30mins. Supe was discarded. Washed with 1mL 70% EtOH, pelleted 16,000g, 4C, 15mins. Supe discarded. MeDIP DNA was resuspended in 100uL of TE (pH = 8.5). Wash samples, containing unmethylated DNA, were resuspended/combined in a total of 100uL TE (pH = 8.5). Samples were spec’d:
Results:
R37: MeDIP DNA = 1.393ug recovery. This is ~13% of the input total gDNA (11.25ug) and is ~28% of the total DNA recovered in the procedure (4.935ug). Unmethylated DNA = 3.542ug total recovery. This is ~31% of the input total gDNA (11.25ug) and is ~72% of the total DNA recovered in the procedure (4.935ug). Total DNA recovery = ~44%.
R51: MeDIP DNA = 1.256ug recovery. This is ~14% of the input total gDNA (8.75ug) and is ~23% of the total DNA recovered in the procedure (5.462ug). Unmethylated DNA = 4.206ug total recovery. This is ~48% of the input total gDNA (8.75ug) and is ~77% of the total DNA recovered in the procedure (5.462ug). Total DNA recovery = ~62%.
There definitely seemed to be a high degree of salt carryover from the procedure, despite the phenol:chloroform treatment and EtOH precipitation. As such, I believe this is the reason that the 260/230 ratios are so out of whack. Possibly explains why the 260/280 ratios for the MeDIP DNA are so high, too?
These results demonstrate what we can expect to recover from this procedure, as well as how much DNA gets lost during processing. MeDIP DNA and unmethylated DNA were stored @ -20C.