Prepping for DNA Extraction
Using PAXgene Tissue DNA Kit (see Kit Handbook) on 2017 Oly gonad tissue fixed in paraffin wax. Tissue was fixed using the PAXgene fixative/stabilizer kit.
Things that I need, in addition to the kit:
• Xylene (we have Xylene Cyanol FF in the chemical storage). NEED XYLENE.
• Homogenizer NEED HOMOGENIZOR. question; do I need to be worried about shearing the DNA? PAXgene advises to “optimize” the homogenization time to not fragment DNA, but that’s one of the next steps that I will do in the workflow…
• Scalpels • Ethanol (96–100%, purity grade p.a.)
• Pipets* (10 µl – 1 ml)
• Sterile, aerosol-barrier, RNase-free pipet tips
• Variable-speed microcentrifuge* capable of attaining 1000–20,000 x g, and
equipped with a rotor for 2 ml microcentrifuge tubes
• Shaker-incubator* capable of incubating at 56°C, 70°C, and 80°C, and shaking at ≥400 rpm, not exceeding 1400 rpm (e.g., Eppendorf® Thermomixer Compact, or equivalent) • Vortex mixer*
• Crushed ice
• Optional: RNase A (100 mg/ml; cat. no. 19101)
The handbook provides instructions for DNA-only or RNA/DNA extraction combined. Do I want RNA in my samples?:
The PAXgene Tissue DNA procedure copurifies DNA and RNA when both are present in the sample. Transcriptionally active tissues such as liver and kidney contain high levels of RNA, which will be copurified. RNA may inhibit some downstream enzymatic reactions, although it does not affect PCR. If RNA-free genomic DNA is required, RNase A should be added to the sample before addition of Buffer TD2, to digest the RNA.
Protocol requires Xylene, which I will purchase unless I hear differently. (Note: we have Xylene Cyanol FF, but I believe that is for staining purposes, not a solvent).
Following Sam and Yaamini’s notebooks, I’ll need Qubit™ dsDNA HS Assay Kit for quantification; I don’t see this in inventory, but are there enough supplies left in the kit for 20 samples?
Same question as 4, for MethylMiner™ Methylated DNA Enrichment Kit
Submitted issue to GitHub with the above questions. Probably won’t be able to actually execute lab work tomorrow after all…