Olympia oyster gDNA that had previously been sonicated and fragmented was enriched for the methylated fragments using the MethylMiner Methylated DNA Enrichment Kit (Invitrogen).
Prepared the following components:
20mL 1x Bind/Wash Buffer (4mL 5x Bind/Wash Buffer + 16mL H2O)
640μL of beads (35μL of beads x 18 samples )
200μL MBD-Biotin Protein (63μL MBD-Biotin Protein + 137μL 1x Bind/Wash Buffer)
Followed the manufacturer’s protocol for input DNA quantities 1μg - 10μg.
Used single fraction, high salt elution.
Neglected to account for the control reaction during initial set up and did not have sufficient quantities of beads to run a control reaction.
The table below provides the individual sample volumes and the volumes of the buffer, beads, H2O for the MBD capture reactions.
Samples listed with “NA” were not processed because they did not fragment during sonication.
| Sample | Volume (μL) | Buffer/Beads (μL) | H2O (μL) | Total (μL) |
| hc1_2B | 75 | 135 | 290 | 500 |
| hc1_4B | 90 | 135 | 275 | 500 |
| hc2_15B | 75 | 135 | 290 | 500 |
| hc2_17 | 75 | 135 | 290 | 500 |
| hc3_1 | 75 | 135 | 290 | 500 |
| hc3_5 | 75 | 135 | 290 | 500 |
| hc3_7 | 70 | 135 | 295 | 500 |
| hc3_9 | NA | NA | NA | NA |
| hc3_10 | 70 | 135 | 295 | 500 |
| hc3_11 | 70 | 135 | 295 | 500 |
| ss2_9B | 190 | 135 | 175 | 500 |
| ss2_14B | 195 | 135 | 170 | 500 |
| ss2_18B | 195 | 135 | 170 | 500 |
| ss3_3B | 190 | 135 | 175 | 500 |
| ss3_4B | NA | NA | NA | NA |
| ss3_14B | 195 | 135 | 170 | 500 |
| ss3_15B | 195 | 135 | 170 | 500 |
| ss3_16B | 195 | 135 | 170 | 500 |
| ss3_20 | 135 | 135 | 230 | 500 |
| ss5_18 | 75 | 135 | 290 | 500 |
Non-captured & wash fractions were pooled into single samples and stored @ -20C.
MBD fraction was EtOH precipitated according to the manufacturer’s protocol and incubate O/N @ -80C.