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
(1) Supernatant after overnight proteinase K incubation…
- the pellet is light and not static, protocol reads to avoid the solid pellet - seems to potentially clog the spin column? Due to its consistency it is very difficult to not suspend the pellet upon pipetting the supernatant
- summary of the issue: low DNA yield likely due to clogged spin column - I have been pipetting ~200ul at this step.
- possible solution: take less volume of the supernatant to conservatively avoid the pellet in future extractions, see if this changes the yield
(2) Cold ethanol
- Note the following change was made for the extraction quantified below
- 100% Ethanol is kept in the ethanol fridge and is added after a 70C incubation period - perhaps the cold temperature after incubation can present issues here
- possible solution (completed today): allocate the necessary volume before extracting to allow the ethanol sample to arrive to room temperature
(3) Warm elution Buffer
- Note the following change was made for the extraction quantified below
- Elution buffer is incubated at 70C for the entire extraction duration and is use twice to elute the DNA, in previous extractions I left this small volume out on the benchtop incubation and spin down as opposed to putting back at 70C IncuShaker
- possible solution (completed today): place the elution buffer back to the 70C IncuShaker as the samples incubate on the benchtop to ensure buffer is at the recommended temperature
Qubit Results
| Sample | DNA (ng/uL) | |
|---|---|---|
| RUN 1 | RUN 2 | |
| Standard 1 | 101.82 | 101.97 |
| Standard 2 | 11081.67 | 10995.73 |
| 2 | 7.89 | 7.79 |
| 18 | 1.55 | 1.52 |
| 25 | 1.18 | 1.20 |
| 26 | 4.28 | 4.33 |
| 27 | 1.11 | 1.15 |