I isolated C.bairdi gDNA yesterday (20200108) and now want to get an idea if it’s any good (i.e. no contaminants, high molecule weight).
I loaded ~100ng (2uL) of the _C.bairdi_ 20102558-2729 gDNA sample, along with two molecular weight markers (see RESULTS below) on a 0.8% agarose, 1x low TAE gel with ethidium bromide. Gel was run for 1.5hrs at 75V.
RESULTS
GeneRuler HighRange Ladder (ThermoFisher):
GeneRuler DNA Ladder Mix (ThermoFisher):
NanoDrop indicates good, clean gDNA (260/230 = 2.12 and 260/280 = 1.99). Although, the NanoPore protocol indicates that the 260/280 should be ~1.75 and values of ~2.0 indicate RNA contamination. Personally, I’d never previously heard that. Interesting!
This DNA was RNase treated, however, the kit I was using was nearly four years old. Maybe the RNase is no longer good (the Proteinase K from that kit certainly wasn’t…).
The gel, on the otherhand, shows less-than-ideal DNA integrity. It’s mostly a degraded smear. This isn’t entirely surprising as the tissue sample had been stored in ethanol at room temperature for nearly a decade. But, the kit I used to isolate the DNA (E.Z.N.A. Mollusc Kit) does have a surprising number of steps where the sample is vortexed (usually a no-no when attempting to obtain intact, high molecular weight DNA).
Regardless, I’ll go ahead and use this for a NanoPore run. This will serve a couple of purposes:
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Familiarize myself with the NanoPore.
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Evaluate whether or not this DNA should be used to submit for PacBio sequencing. If we don’t get long reads from the NanoPore, it would be a waste of money and time to try to get long reads via the PacBio sequencing. In which case, I’d try a different DNA isolation method to try to see if I could obtain higher quality (i.e. intact) gDNA.