Previously, I isolated gDNA from a C.bairdi EtOH-preserved muscle sample (20102558-2729) on 20200108 using the E.Z.N.A. Mollusc DNA Kit (Omega). Although the yields were excellent, the DNA looked completely degraded on a gel and running that DNA on a minION flowcell yielded relatively short reads (which wasn’t terribly surprising).
So, to test out whether or not the degradation was due to the inherent age/preservation method of the sample, I decided to try another kit for gDNA isolation: Quick DNA/RNA MicroPrep Kit (ZymoResearch). I also decided to try three variations of the isolation protocol with very small pieces of tissue (~5 – 10mg):
- “damp”: where the ethanol has not been allowed to dry and homogenized with disposable mortar/pestle 1.5mL tube in 800uL of 1x DNA/RNA Shield.
- “dry”: where the sample is allowed to dry (per the “FFPE Tissue” guidelines) and homogenized with disposable mortar/pestle 1.5mL tube in 800uL of 1x DNA/RNA Shield.
- minced: where the damp sample was minced with a razor blade and incubate in 300uL of 1x DNA/RNA Shield with 30uL PK Digestion Buffer and 15uL of Proteinase K solution at 55oC for 5hrs.
The remainder of the manufacturer’s protocol was followed.
Samples were eluted in 30uL of H2O.
Samples were quantified on the Roberts Lab Qubit 3.0 using 1uL of each sample with the 1x dsDNA High Sensitivity Assay.