Sperm DNA MBD Enrichment Day 4
Re-shearing DNA and checking quality
Based on this issue, I needed to reshear samples and check quality on the BioAnalyzer. The first thing I did was check the concentrations of samples 9, 13, 31, and 63 with the Qubit. The concentrations were smaller than my last readings, which makes sense. My guess is that I didn’t mix the samples properly before pipetting them into the Qubit tubes. I updated the concentrations in this spreadsheet. Based on 31’s concentration, I don’t think it has enough DNA for me to even try MBD with < 1 µg. I decided to drop this sample from my preparation!
While running the Qubit, I sheared samples 6, 12, 48, 57, and 59 for 5 cycles (30 seconds on, 30 seconds off at 4ºC) with the BioRuptor and took out the BioAnalyzer reagents to get to room temmperature. I then diluted the newly sheared samples and the ones I checked with the Qubit (9, 13, 63) to run them on the BioAnalyzer. For all samples except 57, I used a 1:20 dilution (1 µL sample, 19 µL DNAse-free water). Since sample 57 had a higher concentration, I used a 1:100 dilution (1 µL sample, 99 µL DNAse free water) like I did previously. I then prepared the BioAnalyzer chip. The first chip I started to prepare had an issue with the plunger not actually having any pressure applied, so I had to scrap it and use a new chip. At this point I realized I only had two chips left — the one I was about to use, and another one! I made a purchasing request for high sensitivity BioAnalyzer chips and reagents since I couldn’t order the chips separately. Thankfully the plunger worked this time and I was able to prepare my chip.
When I looked at the results, the BioAnalyzer was unable to detect an upper marker for sample 13, but everything else ran well. However, I was unable to toggle between s and bp due to this blip! I took a screenshot of my previous BioAnalyzer run with the x-axis in seconds, then took screenshots of the chip I ran today. I’m hoping I can use the information from the previous run to guess the fragment length for this run of samples. I posted again in this issue to confirm my thinking.
Figure 1. Electropherogram from previous chip.
Figures 2-3. Electropherogram and gel from today’s chip.
Hopefully I’m able to salvage the data from this chip! If I am, then here are my estimates for fragment length:
- 6: Doing something weird…will need to rerun. I remember having trouble figuring out if I actually added any sample to this well.
- 12: Peak around 60 seconds, which might be 200-300 bp
- 48: Peak around 60-100 seconds, which might be 200-600 bp
- 57: Peak around 60 seconds, which might be 200-300 bp
- 13: Peak around 60 seconds, which might be 200-300 bp (but there was also an issue with upper marker detection here)
- 9: Peak around 60-80 seconds, which might be 200-400 bp
- 59: Peak around 60 seconds, which might be 200-300 bp
- 63: Peak around 70 seconds, which might be 300-400 bp
Worst case scenario, I saved the dilutions so I can rerun a chip. I wonder if any other labs have BioAnalyzer chips I can steal until the ones we ordered come in…
- 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
- Investigate low-input MBDBS or WGBS kits for C. gigas gonad samples