Gigas Broodstock DNA Extraction Part 5

Plan for another protocol test

Time to try this again. At lab meeting, we decided to try to get at least 1 ng/µL of DNA because that’s apparently all I would need for a Qiagen assay. I can’t use the Genome Sciences Thermomixer anymore, so I’m improvising. Steven and Sam suggested that I use a heating block to incubate the tubes at the proper temperature. I would keep the tubes on the heating block for a few minutes, then vortex at maximum speed for a few minutes. I would keep alternating until I kept the tubes on the heating block for at least an hour. I might need to extend the incubation time a bit longer to ensure the proteinkinase K digestion happens properly.

Based on my previous trial, I determined that the Tissue Tearor and two minute vortexing with glass beads provided the most consistently high DNA yield. To recap:

  • Tissue Tearor at setting 2 for 20 s (Note: Laura said she used the Tissue Tearor at setting 15 in this lab notebook. She must have used one different than what’s in FSH 213, because there was no setting 15 on the one I used).
  • Glass beads + vortexing for 2 minutes

I’m going to use three tissue samples, split between two tubes (each tube corresponds with each method). I don’t want my tube labels to overlap since I’m going to run these samples and the samples from the previous trial on the Bioanalyzer together. Here are what my tube labels will be:

  • T4-TT
  • T4-V2
  • T5-TT
  • T5-V2
  • T6-TT
  • T6-V2

I’m also not going to eliminate RNA from the samples. I would like to do both bisulfite sequencing and RNA-Seq with each sample, so I want to see if the protocol still works.

Kaitlyn’s helping me out tomorrow, so hopefully this will go smoothly! I’d really like to successfully extract DNA that can then be used for bisulfite sequencing :]

Written on August 30, 2018