Since the previous isolation attempt was unsuccessful (see 20140922), we’re trying a slightly different approach than yesterday.

Today, I will pellet the samples, remove the RNA Later and then proceed with the Quick-gDNA MicroPrep Kit (ZymoResearch) according to the manufacturer’s protocol for Cell Suspensions and Proteinase K Digested Samples.

Isolated gDNA from two C.gigas larvae samples from Katie Latterhos:

B1 400 D6

B6 D00

Pelleted the samples at 10,000g, 5mins, RT. Although no pellets were visible in either sample, the B1 400 D6 sample did have visible cells/debris at the top of the RNA Later after spinning! So, I recovered that portion of the sample for use in the DNA isolation. The B6 D00 sample had no visible debris, nor pellets, so the RNA Later supernatant was removed and discarded.

Both samples were then processed with the Quick-gDNA MicroPrep Kit (ZymoResearch) according to the manufacturer’s protocol for Cell Suspensions and Proteinase K Digested Samples.

Samples were eluted with 10uL of elution buffer and spec’d on a NanoDrop1000 (ThermoFisher). The 2uL used for each sample were recovered from the NanoDrop.

Results:

Note: The B1 400 D6 sample was spec’d twice, due to an error message on the NanoDrop when spec’ing it the first time. Thus, the second entry for B1 400 D6 is the correct value.

Although the B1 400 D6 sample actually yielded gDNA today, the yield is far too low for use in RAD sequencing (need 500ng; B1 400 D6 yielded only ~260ng). Additionally, the quality of the DNA isolated is horrible (OD 260/280 = 0.81).

The B6 D00 did not yield any DNA.

Will let Steven know and see how he wants to proceed.