Isolated DNA from the C.gigas and C.sikamea samples we received from Marinelli Shellfish Company on 20191030 using DNAzol.
Small amounts of mantle tissue were homogenized using disposable mortar and pestles in 1.5mL snap cap tubes using 500uL of DNAzol. After homogenization, an additional 500uL of DNAzol was added to each sample. Samples were mixed gently by inversion. The manufacturer’s protocol was followed with the following notes/changes:
DNA was not spooled; instead it was pelleted per instructions for DNA < 15ug.
Initial DNA pellet wash was performed using 70% DNAzol/30% ethanol
Final pellets were resuspended in 300uL of nuclease-free water.
NOTE: Most pellets were mostly insoluble (i.e. comprised of “junk” that was carried over). After solubilization attempts via pipetting, the insoluble material was pelleted (5,000g, 5mins).
Samples were quantified using the Roberts Lab Qubit 3.0 and the Qubit dsDNA BR Assay Kit (BR = broad range), using 1uL of each sample.
RESULTS
Qubit data (Google Sheets):
| Sample ID | Concentration (ng/uL) |
|---|---|
| C.sikamea_1191-SS-01 | 4.32 |
| C.sikamea_1191-SS-02 | Out of range |
| C.sikamea_1191-SS-03 | 2.74 |
| C.sikamea_1191-SS-04 | 4.34 |
| C.sikamea_1191-SS-05 | 5.84 |
| C.sikamea_1191-SS-06 | 4.34 |
| C.sikamea_1191-SS-07 | 8.18 |
| C.sikamea_1191-SS-08 | 3.6 |
| C.sikamea_1191-SS-09 | 9.36 |
| C.sikamea_1191-SS-10 | 2.92 |
| C.sikamea_1191-SS-11 | 17.8 |
| C.sikamea_1191-SS-12 | 12.4 |
| C.gigas_1191-SS-01 | 7.62 |
| C.gigas_1191-SS-02 | 6.42 |
| C.gigas_1191-SS-03 | 8.24 |
| C.gigas_1191-SS-04 | 6.88 |
| C.gigas_1191-SS-05 | 6.42 |
| C.gigas_1191-SS-06 | 9.7 |
| C.gigas_1191-SS-07 | 9.28 |
| C.gigas_1191-SS-08 | 15.5 |
| C.gigas_1191-SS-09 | 14.5 |
| C.gigas_1191-SS-10 | 10.7 |
| C.gigas_1191-SS-11 | 14.3 |
| C.gigas_1191-SS-12 | 21.8 |
| C.sikamea_CA5SS-01 | 12.3 |
| C.sikamea_CA5SS-02 | 10 |
| C.sikamea_CA5SS-03 | 12.1 |
| C.sikamea_CA5SS-04 | 13.6 |
| C.sikamea_CA5SS-05 | 8.8 |
| C.sikamea_CA5SS-06 | 16.9 |
| C.sikamea_CA5SS-07 | 17.2 |
| C.sikamea_CA5SS-09 | 13.1 |
| C.sikamea_CA5SS-10 | 8.54 |
| C.sikamea_CA5SS-11 | 20.4 |
| C.sikamea_CA5SS-12 | 12 |
Although one of the samples was too low for the broad range assay, I suspect there’s still DNA there. Additionally, since none of these samples are wildly different (i.e. within the same order of magnitude), I probably won’t bother with normalizing the amount of DNA used in the PCR reactions. Will proceed with PCR.