INTRO
Per this GitHub Issue, Steven asked that I select Salvelinus namaycush liver samples from Lean and Siscowet ecotypes for DNA isolation. As part of this project, DNA is being subjected to long read sequencing. A subset of the samples previously sent did not perform well, so need to be replaced.
MATERIALS & METHODS
DNA Isolations
DNA was isolated from five Lean and five Siscowet ecotypes using the Quick-DNA Microprep Kit (ZymoResearch; Cat: D3024), according to the manufacturer’s protocol, with the inclusion of beta-mercaptoethanol in the DNA Lysis Buffer.
Briefly, the liver tissue had been stored @ -20oC in RNAlater. Samples were thawed and the entirety of the sample was transferred to a 1.5mL snap cap tubed. Samples were homogenized using disposable plastic pestles (Fisher Scientific; Cat; K749520-0090). Insolbule debris was pelleted and supernatant was transferred to spin columns. Samples were processed per the manufacturer’s protocol.
DNA was eluted with 50uL of Elution Buffer.
DNA was stored @ -20oC in my “Lake Trout Liver gDNA” box in FTR 209.
DNA Quantification
DNA was quantified using the Qubit 1x dsDNA HS Assay (Invitrogen; Q33231) on the Roberts Lab Qubit 3.0 fluorometer. 1uL of each sample was used.
RESULTS
Qubit data (Google Sheet):
| Sample | Ecoytpe | Concentration(ng/uL) | Volume(uL) | Total(ng) |
|---|---|---|---|---|
| FA091 | Lean | 146 | 40 | 5840 |
| FA092 | Lean | 540 | 40 | 21600 |
| FA093 | Lean | 36.8 | 25 | 920 |
| FA094 | Lean | 214 | 40 | 8560 |
| FA095 | Lean | 253 | 40 | 10120 |
| FA096 | Sisco | 38.8 | 40 | 1552 |
| FA097 | Sisco | 258 | 40 | 10320 |
| FA098 | Sisco | 22.5 | 40 | 900 |
| FA099 | Sisco | 238 | 40 | 9520 |
| FA100 | Sisco | 205 | 40 | 8200 |
Sample FA093 experienced clogging in the column, resulting in low yield.