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):

Sample info 4 Steven etal 112719

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:

Used ThermoFisher Proteinase K (18mg/mL) instead of that supplied by kit. Kit Proteinase K was moldy (!); the kit is nearly four years old…
Lysed tissue for 1hr.
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):

20200108_qubit_crab_gDNA

Yield is very good: ~51ng/uL * 800uL = 40ug

Will run sample on gel and NanoDrop to evalute gDNA integrity/quality.

Sample was stored at -80^oC in:

Rack 6, 4, 1 in C.bairdi gDNA Box #1