Yaamini’s Notebook: Gigas Broodstock DNA Extraction Part 6

Protocol test: Round 4

Today, Kaitlyn fulfilled her dream of getting to do labwork on the weekend (hey, she volunteered to help even after I told her she didn’t need to). As I mentioned in my planning post, we followed the PAXgene protocol to extract DNA from histology blocks with three changes:

  • We had 2 methods for lysis: TissueTearor at setting 1 for 10 seconds, or vortexing with glass beads for two minutes. This is slightly different from the previous TissueTearor methods. I thought I should use a milder TissueTearor setting so I can compare DNA shearing across methods with the Seeb’s BioAnalyzer.
  • We no longer have the Thermomixer. Instead, we’re incubating the tubes on a heat block for 10 minutes, then vortexing at maximum speed for one minute. Total incubation time is still 60 minutes.
  • I’m no longer removing RNA from the DNA I’m extracting. I would like to isolate DNA and RNA from the same samples so I can compare epigenetic and transcriptomic information.

img_9445

Figure 1. Heat block incubation set-up.

I am using four tissue samples — split between two tubes for each method — with eight samples total. Kailyn pre-scraped two samples on Friday (see her lab notebook here) and scraped the other two samples this morning (IDs here). Because the tissue is already preserved in wax, I wanted to see if scraping tissue into a tube beforehand affected DNA yield. Tissue scraping is a rate-limiting step, so being able to scrape out tissue the night beforehand would be a real timesaver!

Method notes

  • Step 9: We incubated the tubes at 37ºC for 25 minutes so the ethanol was fully evaporated
  • Step 12-13: I used the TissueTearor at setting 1 for 10 seconds in the sample. Before use and after samples, I cleaned the TissueTearor in ethanol for 20 seconds, then rinsed in two different batches of DI water for 20 seconds each.
  • Step 15: We incubated the tubes on the heat block at 56ºC for ten minutes, then vortexed for one minute. We repeated this process 6 times for a total incubation time of one hour. There was no gelatinous pellet, so the proteinkinase K digestion must have gone well!
  • Step 17: Skipped!
  • Step 18: The tubes were kept at room temperature for 3 minutes while the heat block reached temperature. We incubated tubes on the heat block at 80ºC for ten minutes, then vortexted for one minute. We repeated this process 6 times for a total incubation time of one hour.
  • Step 27: No DNA came out of the T4-V2 and T6-TT tubes! I think there might have been something clogging the spin column…?
  • Step 28: I made a working solution wiht 2626.8 µL of buffer and 13.2 µL of dye. S1 = 161.34; S2 = 9957.65

Results

Table 1. Initial mass and DNA concentrations for tissue samples processed.

Tube Initial Mass (g) DNA Concentration (ng/µL)
T4-TT 0.0207 3.06
T4-V2 0.0207 N/A
T5-TT 0.0204 7.66
T5-V2 0.0207 N/A
T6-TT 0.0210 N/A
T6-V2 0.0214 2.04
T7-TT 0.0214 N/A
T7-V2 0.0210 N/A

I got DNA, even without a Thermomixer! My yields, however, were pretty inconsistent. They were either 2 ng/µL and above, or N/A. I think part of the problem was the initial mass of tissue: Kaitlyn went above 0.0200 g, maybe because I wasn’t specific about being as close to 0.0200g as possible. The TissueTearor method worked better than the vortexing method. I think the big takeaway here is still variability between tissues.

Going forward

Well, I know the method still works without the Thermomixer! If I’m doing a different bisulfite assay that only needs 1 ng/µL of DNA, I can definitely move forward with my samples. If not, it’s back to testing.

// Please enable JavaScript to view the comments powered by Disqus.

from the responsible grad student https://ift.tt/2NHTImk
via IFTTT

Yaamini’s Notebook: 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 :]

// Please enable JavaScript to view the comments powered by Disqus.

from the responsible grad student https://ift.tt/2LOAtWs
via IFTTT