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.
- 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