I isolated gDNA from ethanol-preserved C.bairdi muscle tissue from sample 20102558-2729 (SPNO-ReferenceNO). This sample was chosen as it had 0 in the SMEAR_result and BCS_PCR_results columns, indicating it should be free of Hematodinium. See the sample spreadsheet linked below for more info.
C.bairdi ethanol-preserved sample sheet from Pam Jensen (GoogleSheets):
I performed four separate isolations using 50mg of tissue. Samples were “ground” * in liquid nitrogen (LN2) with ceramic mortar/pestle and then processed using the E.Z.N.A. Mollusc DNA Kit according to the manufacturer’s protocol, with the following notes/changes:
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Used ThermoFisher Proteinase K (18mg/mL) instead of that supplied by kit. Kit Proteinase K was moldy (!); the kit is nearly four years old…
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Lysed tissue for 1hr.
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Eluted with 200uL for each sample and pooled for final total volume of ~800uL.
Sample was quantified on the Robert Lab Qubit 3.0 using the DNA Broad Range assay, using 1uL of sample.
* The tissue did not powder as one would expect when grinding in liquid nitrogen. Instead, despite all utensils being pre-chilled with LN2 and tissue being actively ground under LN2, the tissue became a frozen paste that adhered to the pestle. I’ve never experienced this before, but I also have not attempted to grind ethanol-preserved tissue in LN2 before.
RESULTS
Qubit data (Google Sheet):
Yield is very good: ~51ng/uL * 800uL = 40ug
Will run sample on gel and NanoDrop to evalute gDNA integrity/quality.
Sample was stored at -80oC in: