Sperm DNA MBD Enrichment Day 6

Final shearing and quality checks

I need to confirm samples 6, 48, and 63 are good to go before I can do MBD enrichment. I made 1:100 dilutions using 1 µL of each sample and 99 µL of DNAse free water, vortexed them to mix, and ran them on the BioAnalyzer with some C. gigas samples.






Figures 1-5. BioAnalyzer results for the whole chip and individual samples

Looking at the electropherograms, 6 may need to be sheared more and 63 should be good to go. The peak is a little lower than I would have liked it to be, but I also vortexed my diluted sample before loading it onto the BioAnalyzer, which could have fragmented the DNA. The electropherogram for 48 doesn’t show any DNA! I added an additonal 1 µL of DNA to my 1:100 dilution, making it a 1:50 dilution. I sheared sample 6 for 2 more cycles (30 seconds on, 30 seconds off) at 4 ºC. I then made a 1:100 dilution and ran another BioAnalyzer chip.



Figure 6-7. Bionanalyzer results for 6 and 48.

Sample 6 looked good, but I still had barely any DNA in 48! I looked in my notes and realized I previously used a 1:20 dilution for sample 48. I made a 1:20 dilution and ran the sample.


Figure 8. Bioanalyzer results for sample 48.

All my samples are now sufficiently sheared! I’ll start MBD enrichment tomorrow.

Going forward

  1. Dilute samples to 25 ng/µL
  2. Prepare reagents for MethylMiner kit (1X Buffer) and label tubes
  3. Process C. virginica sperm samples in two batches with MethylMiner kit
  4. Complete ethanol precipitation with all C. virginica sperm samples
Written on August 11, 2020