Clean and cheap DNA from argophyllus (and other sunflowers)

Argophyllus has a reputation of being a plant it is hard to get DNA from. As a test for a larger project, I did a round of extractions from annuus, petiolaris and argophyllus. I used the modified 3% CTAB method I described before, starting from one fresh, frozen, very young (~1-2 cm long) leaf.

For all species, the CTAB extraction yielded about 50 µl of 200-500 ng/µl solution (10-25 µg in total) of clean (260/230 = 2.05-2.20) genomic DNA, with minimal shearing (see picture).

Ann Pet Arg DNA extraction test

~500 ng of genomic DNA on a 1% gel. Ann = annuus, Pet = petiolaris, Arg = argophyllus (young leaves), Arg M = argopyllus (mature leaves), Arg Z = argophyllus (zirconia beads).

Argophyllus did not seem to behave differently from other sunflowers, despite the fact that young leaves were covered in fluffy, adorable white fur. I also tried to use tissue from mature leaves, which have less dense hair, or a different grinding method with smaller (1 and 2 mm) zirconia beads. Zirconia beads did not do a great job (while regular metal beads produced a nice homogeneous slush), but I went ahead anyway to see if suboptimal grinding would affect negatively the extraction. Both starting with mature tissue  and poor grinding gave slightly less clean (260/230 = 1.9-2.0), less abundant (4-10 µg), but still more than decent DNA.

This CTAB protocol uses beta mercaptoethanol and PVP as reducing agents instead of the DTT in the modified Qiagen protocol Brook used. It could be than using DTT instead would further increase yield, but we run out of it in the lab so I couldn’t test that. Also, it could be that cutting tips as Brook suggested would further reduce DNA degradation. As it is, the DNA would anyway be more than good enough for WGS or GBS libraries (and of course PCR).

This CTAB method can be easily scaled up to 96-well plates, and worked well also with less-than-ideal dried samples from old plants I collected in Utah. Its disadvantages, compared to a Qiagen kit, is that it takes longer (it has a couple of additional steps when compared to a regular CTAB protocol) and involves a chloroform extraction. Its main advantages are that is reliably work with a very different range of starting materials, that you get much more, high quality DNA (shearing has been a problem with Qiagen kits also with other sunflowers in the lab in the past), and that you don’t use a finicky, over-priced kit. An individual Qiagen extraction comes at about 4-5 dollars/sample, or 300+ dollars/plate. Individuals CTAB extractions cost a few cents; plates require special, sturdier tubes, but are probably still one fifth of the cost of a DNeasy plate.