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.

Going forward

  1. Finish shearing and quality checks
  2. Dilute samples to 25 ng/µL
  3. Prepare reagents for MethylMiner kit (1X Buffer) and label tubes
  4. Process C. virginica sperm samples in two batches with MethylMiner kit
  5. Complete ethanol precipitation with all C. virginica sperm samples
Written on August 5, 2020