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…

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
6. Investigate low-input MBDBS or WGBS kits for C. gigas gonad samples