Quick update: 96-well plate CTAB DNA extraction with fresh tissue

When I posted the protocol I have been using for CTAB DNA extraction in 96-well plates, I included results from a few plates I did starting from dried H. anomalus leaves I collected a couple of months earlier in Utah. While they showed that the method worked well enough when starting from “difficult” material, they were not exactly what you’d dream of when you decide to extract DNA, especially if you are starting instead from fresh material.

Here are the results from a plate of extraction I did starting from individual small (1.5-2 cm in length), young leaves from ~3 month-old H. anomalus plants. I collected the leaves directly in 96-well plates (I already put one metal bead in each well), put them on dry ice until I got to the lab (a couple of hours), left them overnight in the -80, and started extracting DNA the day after.


The final volume was 50 microliters, so total yield is for most samples between 10 and 30 micrograms of DNA. These are “real” DNA concentration measured by Qubit. Both average yield and purity are considerably higher than for dry tissue, and they are comparable to what you would get starting with frozen tissue using the single tube protocol (but you save a ton of time). Hope this gets you all more thrilled about 96-well plate DNA extractions 🙂

96-well plates CTAB DNA extraction

When I was working with Arabidopsis, 96-well CTAB DNA extraction was my best friend, and I spent many days extracting away tens of thousands of samples. Good times.

DNA extraction is much less pleasant in sunflower, but since I was reasonably happy with the results of single-tube 3% CTAB DNA extractions, I though I would try to scale it up to a 96-well plate format. Results of earlier attempts, with the participation of Brook and Cris, ranged from inconsistent to disastrous. Things all but improved when I tried again after coming back from Utah with a few hundreds dried samples. Since though the prospect of extracting them all one by one didn’t sound very attractive, I put some more effort into improving the protocol, and now it works quite nicely.

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Momma’s CTAB

Like a few other people in the lab, I’ve been struggling with DNA extractions of late. I’ve tried several methods, including the “columnless” method that a few people are using. But I was having pretty spotty luck. Then, during a bout of methodological soul-searching, I also tried a CTAB protocol that used to be my go-to method. It failed me a few weeks ago, but in a second trial last week, it delivered the goods: loads of very pure, high molecular weight DNA. It might not work for all plants, and there do appear to be situations in which it is not the best option, but if anyone wants to get back to down-home, from-scratch extraction, the way momma used to do it, then give this one a try:

Please note: the results below are for Streptanthus (the plant I’m studying; Brassicaceae); I’m trying it for some Asteraceae right now, and will update the post once I have results.

Momma’s DNA Isolation via CTAB

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