I had that sort of trouble with plants with high concentrations of tanin. I used quiagen kits and at the last step I did 3 elutions in separate tubes the fist one and the second one 100µl and the third one 50µl frequently only the third one (with quite weak DNA) worked. Hope that can help.
Laboratoire Dynamique de la Biodiversité
Universite Paul Sabatier, IV R3
118 Route de Narbonne
31062 Toulouse cedex 4, France
Working on baobabs I've had problems with polysaccharides, but during CTAB also some white cloudy precipitate occuring when adding isopropanol. This precipitate I have yet not resolved what is... However, in my experience, using the dolezel-matab protocol based on nucleus extraction (I reckon you could use ctab instead of matab) solves many problems with secondary compounds, but the yield is relatively low. The reference is Ky et al. (2000) Theor. appl. genet 101: 669-976. I use a slightly modified version that uses 50 ml tubes for the first extraction and thereby lower volumes. I would appreciate to have your answers if possible.
Anders S. Larsen Mimersgade 118, 5.tv. 2200 Copenhagen N
I'm not sure if I can help you, but I have to extract DNA from Hypericum perforatum (with also a lot of secondary components), usually I do a double extraction, or extraction + cleaning. After the normal DNA extraction (Qiagen, NucleoSpin Plant 96 kits,...), I clean the DNA with magnetic beads (Beckman Coulter), and I got good DNA after that. Maybe you could consider this. I hope this would solve your problem. Many greetings and good luck!
Marta Puente Molins
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK)
I have tried two things in these cases.
1) precipitation with 0.5 vol cold 5M NH4Ac: leave for 15 min in fridge.
Spin 10 min., transfer supernatant to new tubes. Precipitate the DNA with 2.5 cold ethanol. Spin 10 min, poor off supernatant. Wash pellet with 70% ethanol, spin down, dry and dissolve in TE (H2O)
2) run out extract on gel. Hope that secondary compounds move with different speed/different direction. Run gel so that BFB is 2 cm or so. Cut out the band, and extract DNA from the gel slice. For example with a gel-extraction kit.
A lot of extra work, although method 1 is doable.
Good luck, Kirsten Wolff
Newcastle Upon Tyne
United Kingdom, NE1 7RU
I had problems with extraction for the species I work on, Gomortega keule (Gomortegaceae). I had oxidation as well as some unknown secondary metabolites (the species is an hallucinogen). I finally came up with the attached protocol which is somewhat based on protocols suggested in the following:
Csaikl UM, Bastian H, Brettschneider R, et al. (1998) Comparative analysis of different DNA extraction protocols: A fast, universal maxi-preparation of high quality plant DNA for genetic evaluation and phylogenetic studies. Plant Molecular Biology Reporter 16, 69-86.
My protocol has just been published in Molecular Ecology Notes early online:
and that paper is attached as well.
Department of Plant Sciences
South Parks Road
Oxford OX1 3RB
Have you tried a treatment with 1:1, 1:2, or 1:3 10% chelex (Bio-Rad) followed by 8-10 min @ 100C (then use supernatant). You might also try 20% in the same ratios.
Works wonders for us with species that are hard to amplify due to inhibitors. We generally treat product amplified from the Qiagen Kits. cheers,
Brant C. Faircloth
Bobwhite Genetics Project
The University of Georgia
I don't have an answer for you, but I would love to hear any suggestions that you get as we are having similar problems with some coral species -- we get great DNA, but it won't amplify and I suspect secondary compounds as well. When I add "test" DNA (some that amplifies) to the reaction, nothing amps, which leads me to believe that something in the reaction is inhibiting the PCR. The one thing that we have tried that worked sometimes is a PEG precipitation -- have you tried that? I am not at the lab at the moment, but can send you a protocol if you would like
All the best,
Mary Alice Coffroth
University of Buffalo Department of Biology
I am not sure if this will work, but I do a lot of scat extractions and subsequentPCR, even though this is a completely different starting product, I am guessing there may be similar secondary compounds inhibiting PCR. Qiagen makes a DNA extraction kit for fecal samples. There are beginning steps where you add your sample to a lysis buffer and then add an "Inhibti-X" tablet which binds the inhibitors. You then spin down the inhibit-X/inhibitors and draw of your supernatant.
Even though Qiagen is quite secretive about the protocol, after these steps everything is the same as the tissue extraction. So basically what you could do is buy the Inhibit-X tablets, and the Buffer ASL, do the InhibitX treatment and then with the supernatant you could do the rest of the extraction. Again, I am not sure if it will work, but it might be something to try...Cheers, Jan
Jan E. Janecka, PhD
Post Doctoral Research Associate
Texas A&M University
I haven’t extracted DNA from grape at all, but I have some colleagues
who have – see below.
Mmm the DNA one is tough. Especially from dried material. The best way to get good DNA from grape is to get fresh or frozen material and do a partial nuclei purification that gets rid of the phenolics before you bust the nuclei. It is probably the phenolics that are the problem but once they are bound on they are there for good normally.
There is a protocol in this paper, but I haven’t looked to see if it’s any different to yours.
Try: Thomas MR, Matsumoto S, Cain P, Scott NS (1993) Repetitive DNA of grapevine: classes present and sequences suitable for cultivar identification. Theor. Appl. Genet. 86:173-180
There is also an enzyme that I don’t know the name of (google search
should find it) which is used for non-specific amplification of DNA that is in very low concentrations that you can then pcr. This might help, but again, I haven’t used it.
I would be interested to know what you are working on. I am working on grape vine rootstock breeding (ie crossing lots of vitis species), but am quite interested in the population genetics of vitis. Also, could you let me know if you do find a successful protocol.
Hope this is of some help.
Dr. Tim Jones
Post doctoral fellow
CSIRO Plant Industry
You have my sympathy - I have had difficulties with many wild plant species, in contrast with domestic food plants which have had all the nasties bred out of them.
One thing not on your list which helped me to crack a particularly difficult one recently (which, as in your case, produced lots of good-looking DNA, but did not want to PCR at all) is DTT (di-thio threitol, "Cleland's Reagent"). I use it at a final concentration of 10mM in the PCR. I now use this stuff with everything, as there does not seem to be any down-side to it.
Other things that have helped at various times are - a tissue rinse buffer step - grind up the material and then suspend it in a large volume (I usually use 30 - 50 ml) of [50mM Tris 100mM NaCl 100mM EDTA 1% PVP pH 7.5], and leave it to soak in the refrigerator for a while (30 min or more - the material will be OK in this buffer indefinitely), then spin down and discard the liquid. In the case of your plants, I would repeat the rinsing two or three times. I do one rinse routinely with everything (perhaps unnecessarily sometimes), and several rinses with things I expect to be difficult. After the rinses, continue as usual (in my case, the digestion buffer is the same as the rinse buffer, with the addition of 0.5% SDS, at 60 Celsius, but you can go to a CTAB-based method, or whatever else you want).
Also, I routinely use the diatomaceous earth clean-up method from the last part of the DNA extraction method described by Gilmore S. P. H. Weston J. A. Thompson 1993 ["A simple, rapid, inexpensive, and widely applicable technique for purifying plant DNA." Australian Systematic Botany 6: 139-148.] However, this is just a home-made version of silica-matrix clean-up that is incorporated into many of the commercially available DNA-extracting kits, so stick with those if you are already using them.
One last suggestion is to try different brands of Taq - changing from Promega to Bioline Taq once solved all my PCR problems with one group of plants that were being very difficult.
Molecular Systematics Laboratory
National Herbarium of NSW
Royal Botanic Gardens, Sydney
I worked with herbivore feces using Promega extraction kits and had some trouble with PCR inhibitors that, according to the literature, were possibly from plant polysacchirides. Using 1 ul of BSA (bovine serum albumin) 0.3ug/ul in a PCR cocktail of 15ul improved our amplification success somewhat, though not entirely. You might try that, play with the concentration maybe and see how it goes. For more information down this line these articles could come in handy:
Monteiro L, Bonnemaison D, Vekris A, Petry KG, Bonnet J, Vidal R, Cabrita J y Mégraud F. (1997). Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model. J Clin Microbiol 35(4):995-998.
Thornton C y Passen S (2004) Inhibition of PCR amplification by phytic acid and treatment of bovine fecal specimens with phytase to reduce inhibition. J. Microbiol. Methods 59: 43-52.
Wilson I. (1997) Inhibition and Facilitation of Nucleic Acid Amplification. App. And Environ. Microbiol. 63(10):3741–3751.
Hope some of that helps,
Universidad Simón Bolívar
Caracas - Venezuela
You can try to clean the compounds out by cleaning your extract i.e. in a Centricon column (I use the size YM-30, you will lose your very small fragments of DNA, but can also get rid of a lot of minerals and compounds) or with an alcohol precipitation (ethyl alcahol and isopropanol will dissolve slightly different compounds, so you could try the isopropanol for the 1st step and ethyl alcohol for the second step) and/or try to overcome the inhibitors in your PCR by adding BSA to your PCR reaction (DTT may help to a lesser extent as well). If you think your DNA is binding to the compounds, you can add PTB to the digestion step of your extraction.
Department of Evolutionary Biology
SE-752 36 Uppsala (Sweden)
I faced similar extraction and amplification problems with Helianthemum sp. I used the traditional CTAB method and Qiagen kits for DNA extraction but unfortunately I got unpurified of a bad quality of DNA and obviously amplification success was 5%. I had to search for an alternative extraction method that remove most of polysaccharides and phenols that inhibit amplification and I found this article:
A simple and efficient method for DNA extraction from grapevine cultivars, Vitis species and Ampelopsis.
Lodhi, Muhammad A., Guang-Ning Ye, Norman F. Weeden and Bruce I. Reisch. 1994. Plant Molecular Biology Reporter 12(1): 6-13
In order to get good quality and quantity of DNA for PCR, I suggest that you take very small amount of leave tissue (0.5 g) and apply only 50 microliter of PVP to 100 ml CTAB SOLUTION.
Hope it will resolve you problem and good Luck,
Eman Soubani (PhD student)
Plant Ecology and Systematic
Dept. of Ecology, Lund University
Ecology Building, 223 62, Lund, Sweden
Qiagen kits typically give pretty clean extractions - it seems unlikely that with so many methods they are all failing to clean the DNA so badly that PCR is inhibited.
Are your extractions green or brown or do they have a large amount of undissolved stuff in them? If extractions are colored, maybe you are starting with too much leaf tissue. Cut back the amount to that recommended by the kit or less.
I suspect that the problem lies in your primers or PCR conditions. Maybe the primers aren't a perfect match to your DNA (as unlikely as it sounds for universal primers) or there is some rearrangement, insertion or deletion in your wild accessions. I hope you are trying your amplification on multiple wild accessions. Also, you might want to try to amplify something different like ITS or a different chloroplast region. If ITS or the other chloroplast regions fail to amplify, then you know that your DNA is bad.
Institute of Arctic Biology
University of Alaska Fairbanks
During my postdoc I dealt with the same problem with tropical woody plants that had been silica gel dried for a number of years. I developed this protocol (reference below, sorry I don't have a pdf) which includes a couple of KEY modifications (PEG in final precip and pretreatment with sorbitol buffer and sarkosyl). If you can't get the pdf, I will be happy to fax you a copy.
California State University, Chico
Modified Polyethylene Glycol DNA Extraction Procedure for Silica Gel-Dried Tropical Woody Plants
Kristina A. Schierenbeck
BioTechniques Vol. 16, No. 3: pp 392-394 (Mar 1994)
Hi there Kate,
Your e-mail was passed to me by a friend. We have used the Qiagen kit on grapevines with moderate success but I now use a much better protocol. I modified it from the attached paper whereby I homogenise 100mg sample in 750ul CTAB buffer using a bead-beater and 2.3mm zirconia beads, heat to 65 degrees for 15 mins and then add the chloroform/IAA directly to the tube. Spin and then take the 300ul supernatant and add 75ul of the 2nd buffer and proceed from there. The yields are OK (about 100ng per ul and 200ul resuspension volume) but plenty for PCR. You could resuspend the pellet in eg 30-50ul if you wanted a higher concn
Let me know if you have any questions
Linnaeus Private Bag 1199 Gisborne
New Zealand Web: www.linnaeus.co.nz
The lab I work for is asked to extract DNA from all sorts of wild plant species, and we have come up with a modification to the Qiagen DNEasy-96 protocol that uses proteinase K (4 uL of a 20 mg/mL solution) to digest secondary compounds. This does not always significantly increase yield, but can do wonders to provide clean DNA that can be restricted or amplified without further problem. I'm attaching our protocol below. We typically flash-freeze fresh tissue and disrupt the frozen samples in Qiagen's mixer mill, but I think you should be able to disrupt dried tissue in the lysis buffer (slower shaking for a longer duration) and then add proteinase K later, as indicated.
I'd be interested in hearing what other techniques you learn about!
Jennifer DeWoody, Biologist
USDA Forest Service, NFGEL
2480 Carson Road, Placerville, CA 95667
We have had some success with the protocol attached, it is a modified CTAB protocol. PDF is original with solution recipes, word.doc is our protocol. I think the reason it works is that it has two extraction phases, first does not break up the plant cells, so you when you dump the LIQUID you are removing nasty leaf compounds with the liquid. The second phase lysis the cells releasing the DNA into solution to use.
Let me know if you have any luck
Chicago Botanic Garden
I work on tanoak, which have lots of tannins and other nasty things. Our lab has had the most success combining a CTAB and phenol-chloroform extraction with the MPBio Geneclean Turbo spin columns, substituting their GNomic Turbo salt solution for the standard salt solution. We pick up with the kit after our phenol-chloroform step, adding the salt solution and proceeding according to their instructions.
I hope this helps! Feel free to email if you'd like more details.
University of California
Berkeley, CA 94270-3114
I have had similar problems with some Begonia species, and had some success with two methods that you are probably already aware of:
1.Wang et al. 1996: An improved procedure for the isolation of nuclear DNA from leaves of wild grapevine dried with silica gel. Plant Mol Biol Rep 14: 369-373.
2. Hanania et al. 2004: An Improved Method for Isolating High-Quality DNA From Vitis vinifera Nuclei. Plant Mol Biol Rep 22: 173*177.
However, the most efficient procedure was to run the same material through two Qiagen DNeasy columns, one after the other (i.e. repeat the extraction procedure on the DNA you elute after the first extraction). With 2X Dneasy extractions I had better results than doing a 1 X CTAB prep followed by 1 X Dneasy column prep.
Also, it can be useful to include the RNase step in your first extraction. If there is any RNA about this helps to get rid of it, leaving more of the column to available to absorb DNA, rather than get clogged up by RNA (at least thats what I think happens!).
Hope this helps in case you have not tried it,
British Antarctic Survey,
Natural Environment Research Council,
High Cross, Madingley Road,
Cambridge, CB3 0ET, UK.