I recently posted a query about plant DNA extractions namely what exactly does CTAB contribute to the process, and what methods do people recommend. Thanks for the replies I received, they were extremely useful. Particularly I wish to thank the following: Donovan Bailey Anders S Larsen Joe Staton Andreas Zipperle Sara Good-Avila Polly Spencer-Vellacott With respect to CTAB itself >>>> I believe that CTAB was developed for use with mucilaginous bacteria to get rid of the very gooey glycoproteins that SDS will not. >>>> CTAB is so popular in plant extractions because early published approaches illusrated its broad utility and so people stuck with it. Most commercially available Plant extraction kits now use protocols much more akin to a typical phage lysis extraction. To the best of my knowledge nobody uses proteinase K in plant extractions - that approach is generally restricted to animal based systems (but it may be useful in some groups). Whether or not to use CTAB or another approach often depends on the quality of DNA you recover from either - they are such different approaches that one will often work well in a particular group of plants while another fails miserably - I would try both and see what works best for you seeds. >>>> Although with regards to Prot K. All of the traditional recipes we have used include CTAB. CTAB binds to the cell wall debris, proteins and polysaccharides in the homogenate: once you perform a phenol-chloroform extraction after the CTAB step, the CTAB/debris/polysaccharide/protein mix is removed. We also include proteinase K to facilitate digestion of the relatively compact tissue in the seeds. >>>>> One other thing I learnt, that may be useful to some of you who might be as naïve with plants as I am I extracted DNA from seeds of Fraxinus excelsior (ash tree). I used a kit (Qiagen) but what may be important (you probably know it already) is that if you are interested in the seed genotype you separate the embryo from the endocarp etc, which might have the maternal genotype. With respect to different method (and here I include names incase anyone needs to chase up anything with the original responder) >>>> We (Donovan Bailey) have recently published a very inexpensive and high throughput method that mimics commercially available kits (who claim to have proprietary approaches - all components of which were actually published many years ago). You can see our paper in molecular ecology notes - http://www.blackwell-synergy.com/doi/abs/10.1111/j. 1471-8286.2006.01549.x >>>> Qiagens plant extracion kits work fine for baobab seeds (scant endosperm) I've experienced, but be aware of the quantity - you easily reduce your yield if adding too much material here! I've seen one protocol where you simply put the required amount of seed into water overnight and extract the decired way (kits or CTAB) afterward, saving the nitrogen. Can't remember the details, but can come back with the protocol next week (Anders Larsen). >>>> I am using microsatellites and I used both, CTAB and a SDS based methods for DNA extraction from seeds. I do not exactly know what CTAB does, but the quality of DNA that I got using a commercial (Sephaglas) and homemade glasmilk is much better with CTAB . Nevertheless, using glasmilk from the really fine-fraction of silica resulted in good quality DNA as well (at least for msats). While CTAB extracted DNA can be stored for at least two years in a -20 freezer this might not be true for SDS. I never used a kit, because our home made method performs pretty good, is streamlined for a 96 well format, can be used with CTAB or SDS and is much cheaper than a kit. If you are interested Elphinstone et al. (2003) Mol Ecol Notes 3, 317 - 320 is the basis for our extraction method and the method itself will be published soon. (Andreas Zipperle) >>>> I've pasted the protocol we are currently using below. This protocol works brilliantly for us - it is fast and gives high yields and very reliable post-PCR. Although it is preferable to remove the seed coat, this isn't always practically feasible or necessary. Currently, we are using this protocol on apple seeds. Modified slightly from Kang et al. 1998. Our (Sara Good-Avila's) protocol for extraction from apple You can start with the whole seed, or the seed - minus the seed coat. Depending on the size of the seed use all or part of it. For apples we use one whole seed/extraction. You can adjust the volume of extraction buffers and final eluate for other seed sizes. - Place the seed in a microcentrifuge tube (1.5 ml). - Add 200 ul of extraction buffer containing proteinase K (50 ug), incubate at 37 C for 1 h. - Grind the seed in the buffer with a glass rod or blue pestle. - Add 200 ul of CTAB solution (2%)(recipe below) - Gently extract using chloroform:isoamyl alcohol (24:1) with 5% phenol. - Centrifuge at 12,000 rpm in microcentrifuge at 4 C for 10 min and transfer the supernatant to new tubes. - Add 2/3 volume isopropanol and incubate the tube at room temperature for 10 min to precipitate DNA. - Centrifuge at 12,000 rpm for 5 min, remove supernatant, wash the DNA pellet with 70% Ethanol, air dry, and resuspend in 30 ul of TE buffer. -Remove RNA by adding 1 ul of RNase (10 mg/ml). RECIPES 1. Extraction buffer: 200 mM Tris-HCl (pH 8.0), 200 mM NaCl, 25 mM EDTA, 0.5%SDS. 2. 2_ CTAB (cetyltrimethylammonium bromide) solution: 2% CTAB(w/v), 100 mM Tris-HCl (pH 8.0), 20 mM EDTA (pH 8.0), 1.4M NaCl, 1% PVP polyvinylpyrrolidone) Mr 40,000. mtpgilbert@gmail.com