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.
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!
- 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
Table 1. Initial mass and DNA concentrations for tissue samples processed.
|Tube||Initial Mass (g)||DNA Concentration (ng/µL)|
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.
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.