Thanks Brjmohan, I 'd like to see have you ever tested this method in your experiments?

I'm going to do dna extraction for woody plant too..thanks alot!

For grapevine, DNA extraction is also quite tricky because of polyphenols and polysaccharides. I know that the method they used in this paper is suitable: http://link.springer.com/article/10.1007/s12033-013-9675-3#page-1 (it's not open access, but you can still see the method on the second page of the preview). Hope this can help. I think CTAB remains the best way, with minor modifications.

Do you want to extract from leaves or stems?

Hi,

We have routinely extracted DNA from a range of plants that are high in polyphenols. The most robust method is based on CTAB extraction. The key addidtions to the protocols are PVP and PVPP at around 2.5% w/v and 2-BME up to 5% v/v. These additions will assist in binding polphenols and prevention of any oxidation reactions promoted by PPO enzymes. Probably the most important step is to avoid using any phenol in the extractionns until after the first precipitation step. We have also found the use of phaselock gel/tubes (5-prime) greatly improve the extractions as they form a solid barrier between the organic and inorganic phases in the extractions with the protein trapped below the solid layer allowing you much better yeild recovery and much better purity.....worth the small expense.

For our grapevine work we have found theat M&N plant minispin DNA kits work very well for leaves. However a word of caution....while these kits are great if they work we have found them to be very tissue and plant specific.

Extraction with CTAB and PVPP .(PVPP bind the polyphenols).

PVP and PVPP definitely help, yes!

Dear Brijmohan,

Thanks for the article with the protocol. We have had many problems with DNA extraction of same Brazilian trees, even by using the CTAB with PVP and PVPP. Hopefuly, the CTAB-activated charcoal helps to solve the problems. We will keep you inform about our results.

You can also try this method it's been successful in most woody plants with high polyphenol content :

http://world-food.net/a-simple-and-rapid-method-for-isolation-of-dna-from-imbibed-embryos-of-parkia-timoriana-dc-merr-for-pcr-analysis/

I can provide you a copy if you can send a mail to me: [email protected]

Try using a hot boric acid method, derived from the original CETAB methodology.

The C-TAB method has also provided the best results for nucleic acid extractions from Persea americana (avocado)

Sharma et al. (2013). Conventional and high throughput DNA extraction for PCR amplification using wide range of tissues: seeds, tubers, leaves and infected tissues. African Journal of Biotechnology. 12(15): 1894-1901.

Best wishes.

I have extracted DNA from highly perennial forest trees with excessive loads of phenolics and polysaccharide. Find the following research article

Saha D. (2010). Journal of Non Timber Forest products. 17(4): 395–400.

You can also replace CTAB with SDS and you will be amazed at the results

We have had very good results using

Bellstedt, D.U., M.D. Pirie, J.C. Visser, M.J. de Villiers & B. Gehrke (2010) A rapid and inexpensive method for the direct PCR amplification of DNA from plants. American Journal of Botany 97: E65-E68. doi:10.3732/ajb.1000181

also with samples that have high percentages of phenol and polysacharid (http://www.amjbot.org/content/97/7/e65.full)

I tested different DNA extraction method from woody tissues and now I use the MACHEREY-NAGEL NucleoSpin Plant II

You can apply following methods for tree species.

Gupta AK, Harish, Rai MK, Phulwaria M, Shekhawat NS (2011) Isolation of genomic DNA suitable for community analysis from mature trees adapted to arid environment. Gene 487:156-159

Our method involves (i) washing the sample twice with Triton buffer (2%) then (ii) isolation of gDNA by modified-CTAB (cetyl trimethyl ammonium bromide) method employing a high concentration (4%) of PVP (Polyvinylpyrrolidone) and 50 mM ascorbic acid, and (iii) purification of this CTAB-isolated gDNA by spin column.

gDNA isolated by modified CTAB or spin-column alone were not found suitable for PCR amplification. The Triton washing step is essential.

We use MACHEREY-NAGEL NucleoSpin Plant II or Qiagen for extraction from Eucalyptus leaves and wood cores for DNA finger printing analysis.

ere is the protocol we use for DNA extraction from olive leaves (Olea europaea):

i think CTAB will yield the desired results

Use CTAB method, in addition crush your sample with PVPP or add PVPP after crushing during homogenization in extraction buffer containing CTAB. PVPP will remove phenolics.

I recently worked with DNA isolation of Ficus, Bamboo and Ziziphus plants using CTAB method (with 2% PVP). DNA amount obtained was good but 230/260 ration was problem (2.0). PCR was run with 6-10ng of diluted DNA samples. It worked well.

So my suggestion is; If you have already isolated DNA with undesired 260/230 ratio, use diluted samples for PCR. Right from 2ng to 20ng. You may also treat your DNA sample with activated charcoal (add pinch of activated charcoal, shake for 10 min to 60 min; time varies with sample to sample) to remove contamination of phenolics.

I have successfully used the method of Aljanabi et al to extract gDNA from Norway spruce somatic embryos and Arabidopsis seeds both high in polythenols and polysaccharides.

Article An Improved and Rapid Protocol for the Isolation of Polysacc...

I agree that modify CTAB and PVPP is suggested. But the most import thing will be concern what part you uas to extract the gDNA. I will suggest the youngest part such as meristem wish content lest second metaboism material will more easy to clean up the DNA you extract.

You can try using a standard DNA Extraction with an adapted CTAB method as follows: - Use 4 ml of sample solution starting from 20mg sample - Mix with 2 ml of CTAB buffer (100 mM Tris–HCl pH 8.0, 1.4 M NaCl, 20 mM EDTA, 2% w/v CTAB, 0.2% v/v b-mercaptoethanol, 1% DTT w/v, added before the use) - Incubate at 65 C for 30 min, with continuous shaking. - After cooling at room temperature, the mix is added with an equal volume of chloroform isoamyl alcohol (24:1, v/v) - Centrifuge at 14,000g for 20 min.- The upper phase is then transferred in a fresh tube, - Mix with one volume of cold 2-propanol- Centrifuge at 14,000g for 30 min at 4 C. - The pellet is washed two times with 70% (v/v) ethanol and suspended in 60 ul of water. - Use distilled water to generate a negative control

Hello, I have a similar problem! I have to extract a clean DNA in high amount from leaves of a tree. One important thing could be the ratio between amount of grounded leaves and ctab buffer: if in your falcon powder arrives for example to 5 ml, split in almost two aliquotes with 25 ml ctab buffer each. Before continuing with two (instead of one) chloroform extractions, I centrifuged the tubes to remove all undigested powder.

I have also found this paper that could be useful: A novel and efficient protocol for the isolation of genomic DNA from mulberry (Morus L.) . Google this title and you will get the pdf. I did not try it yet but I am going to do. For the rest, I have posted a similar question, so I am waiting for suggestions too :)

We have worked with coffee plants which have high phenol and polysacharide content. We have developed a protocol and published it.

A simple method of DNA extraction from coffee seeds suitable for PCR analysis

Manoj Kumar Mishra* et al. African Journal of Biotechnology Vol. 7 (4), pp. 409-413, 19 February, 2008

You can refer to this article which may be useful.

We routinely extract DNA from Eucalyptus wood samples for DNA fingerprinting using the Nucleospin kit from Machery-Nagel with the following modifications:

1. We grind the sample using an IKA tube mill and liquid nitrogen

2. Incubate at 65 C for 30 mins at the protein precipitation step

3. Add an additional wash step with buffer PW2

4. Incubate at 70 C for 10 mins at the elution step

5. Elute in 50 ul elution buffer, twice

This yields high quality DNA but the concentration is a bit lower than we would prefer, so we evaporate using a miVac DNA concentrator and then resuspend in 50 ul low TE buffer. The DNA concentration and quality is then up to standard for Illumina SNP genotyping, sequencing with BGI and other more routine applications

'Alkaline PVPP method' can extract high concentration of DNA from leaves of any plant:

Large-scale extraction of pure DNA from mature leaves of Cyclamen persicum Mill. and other recalcitrant plants with alkaline polyvinylpolypyrrolidone (PVPP)

http://www.sciencedirect.com/science?_ob=ArticleListURL&_method=list&_ArticleListID=-1032114230&_sort=r&_st=13&view=c&md5=ec168676af56d24df069f41c8c8829fb&searchtype=a

Please incresase NaCl concentration to 4 M, from the original buffer in this paper.

After phenol-chloroform treatment, dilute with the same volume of water, to prevent NaCl precipitation, before isopropanol or EtOH treatment.

DNA extract had better be checked by agarose gel electrophoresis, rather than UV absorbance (A260 value).

Extraction buffer with CTAB, PVP and B-mercaptoethanol yield sufficient and high quality DNA from woody plants such as coffee with high phenolics and polysacarids. We are successful in isolating high quality DNA using this method. Please refer to our papers . It may be helpful to you. We have used leaf samples as well as seed samples.

1.MOLECULAR IDENTIFICATION AND GENETIC RELATIONSHIPS AMONG COFFEE SPECIES (COFFEA L) INFERRED FROM ISSR AND SRAP MARKER ANALYSES

2.Genetic molecular analysis of Coffea arabica (Rubiaceae) hybrids using SRAP markers 

A simple method of DNA extraction from coffee seeds suitable for PCR analysis

You may find some idea from this research article; genomic DNA isolation was standardized from very hardwood tree samples loaded with tonnes of phenolic substances and polysaccharides as well. Best of luck.