Objective and summary:
- Measure DNA concentrations of DNA extracted from F0 adult Bay scallop adductor tissue (dissected and extracted in early January 2022)
- DNA was extracted using the OMEGA EZNA Tissue kit protocol here
- Ran Qubit dsDNA BR kit following standard protocol thermosci online pdf
Changes/Recommendations to the OMGEA Extraction protocol!
NOTE: The previous extractions in late December 2021 and January 2022 resulted in low DNA yield via qubit (review table in F0 Aropecten Qubit 20220104) therefore I made a few changes and paid close attention to particular aspects of the protocol
Supernatant after overnight proteinase K incubation…
- used spin columns after the overnight incubation to potentiallu catch the loose tissue pellet. This worked - spin column was discarded and I took the 200ul sample
- the rationale here was that I may have been taking p some of the pellet and it clogged the spin column downstream, decreasing yield, however this did not increase our yield!
Qubit Results
| Sample | DNA (ng/uL) | |
|---|---|---|
| RUN 1 | RUN 2 | |
| Standard 1 | 92.66 | 95.12 |
| Standard 2 | 10572.79 | 10362.27 |
| 24 | 2.03 | 1.95 |
| 19 | 3.79 | 3.50 |
| 17 | 3.23 | 3.28 |
| 5 | 1.32 | 1.25 |
| 9 | 10.9 | 11.1 |
Moving forward…
- as we see here I made several adjustments to no avail (DNA yield still low)
- next extractions I will do the following:
(1) Use more tissue
- the protocol (OMEGE EZNA) asks for 30 mg of tissue and I have been following this - double the tissue and proteinase K to see if this increases DNA
(2) Decrease volume of Elution Buffer
- use 2 elutions at 50 ul each as opposed to 100 ul. This should at least double our possible DNA concentration - our main bottleneck for library prep is concentration of DNA and not total DNA so highly concentrated low volume is perfect