Objective and summary:
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Run ethidium bromide gel electrophoresis for post-library prep samples F1 Juvenile Bay Scallop 8C 101 and 102
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Library prep was (for the first time thus far) the full ‘modified Baym’ protocol (site here - to be written up in a notebook post)
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Note: ran with a 1% agarose gel (0.75 g agargose 75 mL TAE) for 100 mV for 60 minutes
Gel Electrophoresis
Gel map is as follows:
| well | 1 | 2 | 3 |
|---|---|---|---|
| samp | L | 8C 101 | 8C 102 |
Gel image
- we see A good smear for bands in wells 2 and 3 for ~800-1000 bp (post library prep from today)**
Qubit quanitification
dsDNA BR kit
| Sample | DNA (ng/uL) | |
|---|---|---|
| RUN 1 | RUN 2 | |
| Standard | 60.12 | 59.54 |
| Standard | 5533.02 | 5390.27 |
| 8C 101 | 18.0 | 18.4 |
| 8C 102 | 12.5 | 12.7 |
Summary
Takeaways from using the Modified Baym protocol
- 2.5 ng of input DNA yielded 32 ul of a final volume averaged ~15 ng/ul (~480 ng total DNA!) - this protocol worked great, better than Illumina DNA Prep recommendations!
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aside from the major aspects changed from Ilumina DNA Prep (tagmentation, amplification, clean up reagents, volumnes, input DNA, etc.) I inserted a ‘hold’ temp in the thermocycler at the same temp to the start for each program (i.e. 72C start of the PCR program, set a ‘hold’ at 72C prior and begin this program ~5 minutes before loading samples); perhaps this made the difference in our PCR?
- next step: trudge through extractions, I am ready to get this going in bulk. waiting on plate centrifuge to ensure proper protocols and spin downs of indices