Sperm DNA MBD Enrichment Day 5
Continuing shearing and quality checks
We got BioAnalyzer chips on Friday, so it’s time to run more chips! I took the dilutions I made previously (1:100 for sample 57, 1:20 for all other samples) and loaded them onto the chip. My run (thankfully) was successful!
Figures 1-2. Electropherogram and gel for resheared samples
Most of my samples have peaks around 300-400 bp, which is good for the MethylMiner kit. Samples 6 and 63 have peaks around 1000 bp, so I ran them for five cycles (30 seconds on, 30 seconds off). I wasn’t sure about sample 12, which has a significant amount of DNA that’s sheared correctly but also some DNA that’s longer. I posted this issue to get Sam’s opinion about reshearing. I figured I’d hold off on running samples 63 and 6 on the BioAnalyzer until 1) I confirm if I need to shear more samples and 2) I conduct an isopropanol precipitation with C. gigas gonad DNA samples so I can conserve BioAnalyzer chips.
- Finish shearing and quality checks
- Dilute samples to 25 ng/µL
- Prepare reagents for MethylMiner kit (1X Buffer) and label tubes
- Process C. virginica sperm samples in two batches with MethylMiner kit
- Complete ethanol precipitation with all C. virginica sperm samples