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.
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:
Familiarize myself with the NanoPore.
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.