More CTAB!

Since I had as well troubles to get DNA extractions working with the protocols going around in the lab, I tried out a few published protocols (+ the regular CTAB extraction I used for Arabidopsis), picked the one that performed best in my hands and played around with it a bit.

Here’s the result: Sunflower CTAB DNA extraction protocol v1.2

With respect to traditional CTAB methods, it includes an initial wash of the ground tissue to remove some of the more obvious garbage, uses a more energetic 3% CTAB solution, and includes a second ethanol precipitation. It is therefore a bit longer than the protocol Dylan suggested, and I guess in the end it depends on how problematic the tissue you are working with is.

I used this protocol on both freshly collected leaves (anomalus, annuus), tissue that had been stored for a while in the -80 (anomalus, deserticola) and dried tissue (anomalus), and it always performed very well. For the latter in particular I didn’t have high hopes, since the phyllaries I used had abundant sticky resin-like stuff on/in them, but most of the samples I processed gave very reasonable amounts of clean DNA. I should mention that in this case I probably used way too much tissue (being spoiled with Arabidopsis, where it really doesn’t matter much). Given what Dylan, Dan and Greg B noticed, starting from less tissue might give even better results (I am trying that right now). Also, if you start from dried leaves things should be much easier.

A few methodological notes:

– initial wash: I can’t recommend it enough. It really helps, especially for difficult tissues. From some the dried anomalus samples it would get rid of a lot of gooey stuff. You might be able to skip it if you start from easier tissue, but it doesn’t seem to affect the final yield, and it gives much more consistently clean DNA.

– RNAse treatment: I like to add RNAse to the CTAB buffer. In my experience the RNAse doesn’t really mind the beta-mercaptoethanol, and that way you are sure to get rid of it with the chloroform:isoamlylacohol extraction. I didn’t notice any difference in the output DNA when I tried instead to add the RNAse to the TE buffer, when resuspending the pellet after the first ethanol precipitation. Anyway, it’s up to your personal preferences. Ups! It appears I was wrong 🙂 Dan found still a bit of RNA in his preps. I guess there is too much beta-mercaptoethanol in that CTAB solution. So, add the RNAse when you resuspend the DNA. My bad. The protocol has been modified accordingly.

– second ethanol precipitation: this is not strictly necessary, at least if you start from a friendly tissue, but it does improve marginally the quality of the final DNA, and you lose only a minimal amount of it. I didn’t try skipping this step when starting from dry tissue. If you skip it, you have to move the RNAse treatment to a previous step.

– yield: for fresh or kept-in-the-freezer tissue, I had average yields of about 8-15 μg (Qubit readings; at least in my experience, the Nanodrop seems not to be the best method for quantifying DNA – according to it some of my samples contained more than 2 mg of DNA…). For dry tissue yield is a bit lower, around 5-12 μg. As mention earlier, that might also be due to me starting with too much material.

I hope this is of some help. I have plenty of buffers, so if you’d like to try it without too much commitment I’d be happy to share them. And if you do try this protocol, it would be useful to have your feedback.

3 thoughts on “More CTAB!

    • Sorry for the late reply. I started from 1 small frozen leaf, I never was too much into weighing tissue…I did last week some more extractions from 1-2 very young frozen leaves (maybe about 2 cm long) from adult H. anomalus plants, and got from 50 to up to 120 (!) μg of clean DNA.
      The idea behind using young leaves is that most cell division occurs when the leaf is really tiny (well, at least that’s the case for Arabidopsis, but I guess it’s the same in most plants), and afterward you have mostly cell expansion (which comes with some endoreduplication, but unlikely enough to compensate for the increased volume). The bottom line is that using very young leaves you start with a higher ratio of DNA to other cellular garbage (in particular I guess secondary metabolites that might accumulate later on in the expanded vacuole). Or maybe not, but it does kind of make sense to me, and I did get much more DNA.

  1. As I mentioned in the corresponding blog post, at least for 96-well plates CTAB DNA extraction DNA purity is greatly improved by reducing the centrifugation time in the chloroform extraction step and precipitating the DNA for no more than 20-30 minutes in isopropanol at -20°C afterwards. I haven’t checked if this would make any difference for the single tube protocol as well, but if you are having DNA purity problems it might be worth giving it a try.

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