Hi,

What you are experiencing is not uncommon in the plant world as the post above attest to. It is most likely your contaimination is due to the presence of polyphenols and quite probably polyphenol oxidases. The combination of the two lead to the production of reactiuve quinnones in the presence of oxygen. These reactiive compounds then interact with the DNA and RNA to both decrease yeilds in most commonnly used extraction methodologies as well as inhibiting downstream enzyme based manipulations such as PCR.

All of the suggestions above will work to a greater or lesser degree. The addition of PVP and PVPP as well as increasing 2BME concentrations to between 5-10% works well. I have also found that removing phenol from the initial extraction (using only chloroform + isoamyl alchohol) also greatly enhances the yeild and purity of nucleic acids. Many of the commercial kits contain guanidinium based extraction reagents rather than CTAB and these may also cause you some issues. If you use the more traditional organic solvent extraction methodology I would strongly recomend using PhaseLock tubes from 5-prime - these act to provide a solid gel like interface between the organic and aqueous phases greatly enhancing your yeild and purity. THey are not expensive and the quality gains far outweigh the initial cost.

A word of caution using column based clean-up kits - we have found that the quality of recovered nucleic acid is excellent the yeilds recovered are often quite low indicating quite significant losses. Also in my experience once you have oxidative processes at work in your extraction there is very little you can do to improve the quality of the resulting DNA/RNA....best to get the extraction right in the first place.

Good Luck

Suzanne Kennedy (of MO Bio) would be a boon in the way of addressing your question:

http://sandiego.scienceonline.com/2012/10/11/interview-with-scienceonline-panelist-suzanne-kennedy-mo-bio-blogger/

Hello,

I had the same problems with contaminants, my DO ratio 260/230 was lower than 1 (which mean Guanidine, thyocyanate and phenol contamination). I tried a purification on PVPP colons but it doesn't work (loss of RNA)

The better solution I found is to add a small quantity of PVPP directly to your plant tissues after adding the extraction Buffer (RLT or RLC for Quiagen Kit)

Good luck

PVPP could help if you add it to the first extraction step. Also a chloroform:isoamyl (25:1) extraction.

I have attached a protocol we used for DNA extraction from difficult tissues (high in polyphenolics, carbohydrates and other secondary metabolites). Good luck!

How about adding RNase A and Potease K

We call T4 gene 32 protein "magic protein" in our lab, as it stabilises Taq DNA polymerase and removes many PCR inhibitors. As a result it has made many a difficult or dirty extraction product into a beautiful clean PCR product. It is quite expensive, but you only add 0.125ul to each 25ul reaction, and it seems to work on a variety of inhibitors:

http://aem.asm.org/content/62/3/1102.full.pdf

3-4 years ago I had same problem, I used a simple method which was published in Plant Molecular Biology Reporter (Aljanabi SM, Forget L. and Dookun A, 1999, 17:1-8). It was so effecient to remove of PCR inhibitors such as the polysaccaride and polyphenols. I think this method can solve your problem, you should try.

Good luck

Semra Hasancebi

Hi, we found that simply increasing the Beta mercaptho ethanol concentrations to 5 - 10% in the first CTAB extraction step eliminated most PCR inhibitors. If this does not work I will clean up the DNA with an ion exchange column. This should get rid of almost all contaminants.

Hi, you can try with phenol:choloform:isoamyl alcohol (25:24:1) instead phenol:choloform, include PVPP in CTAB buffer and to precipite the DNA with isopropanol (half volume some times is not sufficient, you can use a volume without problems). Another option is use a Sephadex column, and pass a diluted DNA by the column by centrifugation and collect it in a fresh tube, then, concentrate the DNA by evaporation or reprecipitation.

Good luck!

These articles would be helpful:

http://www.hartnell.cc.ca.us/faculty/jhughey/Files/dnaextractionburnedbones.pdf

http://jcm.asm.org/content/39/10/3750.full

This might help. I used extensively to get RNA from pine tissue. No kits. Stinky. Not sure if you are getting DNA or RNA. This may be adaptable to DNA.

I think that problems are not in the methods or sophisticated protocol of extraction DNA. Maybe the problems are in the condition or standarization of this methods in your lab. I am refering that all that your are used in the purification. You will need establish a control of quality that garnanted that all is ready for used in one purification. And after I recommend checking a simple and easy protocol for extraction DNA to use in AFLP.

Hello good day!

I recommend the use of PVP (polyvinylpyrrolidone) is used precisely for these cases.

As partuicular, I've had good results with this product.

You could still use the 2% CTAB solution and add about 50-80 mg of PVP during grinding the tissue to be removed, then add your CTAB 2%. the end of its nucleic acid extraction quality improve its relations 260/280 and 260/230, getting a good bend in his Nanodrop (or depending to use to measure)

You can also do a double wash after having precipitated the nucleic acid with 70% ethanol (this washing improved the quality)

On the other hand, also gives good resultandos making dilutions, could use 1/30 or 1/50 or nucleic acid may dilute your taking it to a concentration of 50 ng / µL

All this will help a lot and if your subsequent nucleic acid required for PCR, nucleic acid will have a good quality to amplify.

Good Luck!

Hi, and welcome to the PCR problems world!

Despite the extraction method you use (but kits gave you a better result) many people are use a new aditive in the PCR reaction that is thought to remove the inhibitors. It is a Trehalose-Based Additive, and you can find the recipe in the link below:

http://www.bioone.org/doi/abs/10.3732/apps.1200236

________________________________________________

Applications in Plant Sciences 1(1):1200236. 2013

doi: http://dx.doi.org/10.3732/apps.1200236

Enhancing PCR Amplification of DNA from Recalcitrant Plant Specimens Using a Trehalose-Based Additive

Tharangamala Samarakoon, Shiao Y. Wang, and Mac H. Alford

_________________________________________________

Good luck!

Hi, I had the same problem and simply solved it by diluting my samples. But I dunno if it would work in your case or not :)

Good luck

Hi,

What you are experiencing is not uncommon in the plant world as the post above attest to. It is most likely your contaimination is due to the presence of polyphenols and quite probably polyphenol oxidases. The combination of the two lead to the production of reactiuve quinnones in the presence of oxygen. These reactiive compounds then interact with the DNA and RNA to both decrease yeilds in most commonnly used extraction methodologies as well as inhibiting downstream enzyme based manipulations such as PCR.

All of the suggestions above will work to a greater or lesser degree. The addition of PVP and PVPP as well as increasing 2BME concentrations to between 5-10% works well. I have also found that removing phenol from the initial extraction (using only chloroform + isoamyl alchohol) also greatly enhances the yeild and purity of nucleic acids. Many of the commercial kits contain guanidinium based extraction reagents rather than CTAB and these may also cause you some issues. If you use the more traditional organic solvent extraction methodology I would strongly recomend using PhaseLock tubes from 5-prime - these act to provide a solid gel like interface between the organic and aqueous phases greatly enhancing your yeild and purity. THey are not expensive and the quality gains far outweigh the initial cost.

A word of caution using column based clean-up kits - we have found that the quality of recovered nucleic acid is excellent the yeilds recovered are often quite low indicating quite significant losses. Also in my experience once you have oxidative processes at work in your extraction there is very little you can do to improve the quality of the resulting DNA/RNA....best to get the extraction right in the first place.

Good Luck

When working with tough, fibrous grass leaf tissue in the past, I found adding PVP to the PCR reaction worked well. As others have mentioned, you can add it during the extraction step, but I had already done the extractions and had no material left.

See this paper:

Koonjul et al. (1999) 'Inclusion of polyvinylpyrrolidone in the polymerase chain reaction reverses the inhibitory effects of polyphenolic contamination of RNA'. Nucleic Acids Research 27(3):915-916.

It'd be specific to the tissue you're using though - obviously it will depend what your inhibitors are.

Other than the optimization of method for obtaining DNA of high quality,you can increase the PCR efficiency by using some PCR enhancers against different inhibitors like DMSO, BSA, Betaine ,Formamide,PEG all are commonly used in the labs or simply dilution series of your DNA can give better amplification

Hi, you can use Amicon Ultra centrifugal filter, it will allows you, not only purify your DNA also concentrate it. Also I suggest make dilutions of DNA in your PCR.

Dear Joshua,

To remove any type of contaminants and purify DNA, the best way is to re-precipitate using 3M NaOAc and 95% Ethanol or Isopropanol. Then wash 2 times with 70% Ethanol, air drying and finally dissolve in 1XTE or Pure water. This could help if you have large amounts of DNA initially.

But if you have less DNA to start with, I would highly recommend using a very quick and easy method, which involves using Ampure XP Magnetic beads for PCR clean -up from Beckman Coulter. The method is ultrafast and you will have a recovery of more than 95% of the starting material, which is a way higher efficiency than any other method.

https://www.beckmancoulter.com/wsrportal/wsr/research-and-discovery/products-and-services/nucleic-acid-sample-preparation/agencourt-ampure-xp-pcr-purification/index.htm

Cheers,

Sudip

You can try to use a DNA kit for Stool...are expensive and need hands time, but you'll obtain good DNA quality

The trouble with plant tissue like cambium is poor ratio between DNA and contaminants. Dilution may not be a good option since you decrease concenttration of your precious DNA. We are using precipitation of polyphenolic contaminants with a high salt ammonium acetate (fnal, 2.5 M NH4COO) following the procedure: add 10M NH4COOH (ammonium acetate) to the concentration 2.5M (roughly one third volume of the DNa sample), leave 30 min at 4C, centrifuge at 10000 x g for 5 Min. The polyphenolic compounds should be found in the pellet, your DNA remains in the supernatant. Then,precipitate DNA from the supernatant with 2.5 volume abs. EtOH, wash with 70% EtOH, resuspend DNA in EB buffer (approx 30% the volume before purification.Hope it works.Ales

Washing with HEPES buffer after grinding and prior to Starting DNA extraction with any of the protocols will help. Prepare a solution containing 0.1M HEPES buffer, PVP, L-ascorbic acid and 2-mercaptoethanol and then add 1000ul of the mix, centrifuge for few minutes and remove the liquid part. You can then continue with any of the extraction protocol you want.

All the best

PCR inhibitors can be removed by adding more of DNA polymerase or

DNA template may be diluted or add additives to the PCR r eaction like BSA or Betaine or NaOH treatment or addition of Aluminium Ammonium Phosphate or separation step prior to PCR with Centricon-100 or Micro -100 filters.

I would suggest rather using a polymerase that are resistant to the normal inhibitors. We are excited about the Kapa3g product from Kapa biosystems, http://www.kapabiosystems.com/products/name/kapa3g-plant-pcr-kits, but there are others available. This way you should hopefully not have to worry about this issue.

Hi Joshua. Usually to remove any type of contaminants by re-precipitation of the DNA using 0.5 volumen of 7.5M AmmoniumAc and 3 vol of Ethanol. After centrifugation I dissolved in 1XTE or Pure water.

Hi Joshua.

I have had problems with very viscous DNA.

I tried many protocols and find this one to work very well yielding high molecular DNA (see below). I skipped the Qiagen genomic tip in this extraction.

Re-precipitation with sodium acetate and ethanol is not done with 3M NaAc as mentioned above, but with 0.1M final concentration. You usually prepare 3M NaAc and add 1/10 vol to your solved DNA, then add 3vol pure EtOH. It is always helpful and cleaning to do this.

No matter how you precipitate, the washing step with 70% EtOH is essential, values get better if you shake the tube with the DNA pellet and the 70% EtOH a little, just so the pellet comes off the tube wall, ten centrifuge again for 5 min at ~11000g.

Dissolving the pellet works best if you add your solvent (H2O or TE) and just let it stand at room temperature for a while. Doesnt harm the DNA.

Good luck.

Ulrike

We have been using the PowerPlant Pro DNA extraction kit from MO BIO. (http://www.mobio.com/plant-dna-isolation/powerplant-pro-dna-isolation-kit.html). These have been optimized for poly-phenols and polysaccharides removal. We have been getting very good results with the subsequent amplification of both fungal and plant DNA.

We get rid of polysaccharide contamination by a salt wash. Most polysaccharides dissolve in concentrated salt solutions while RNA does not. Pellet the RNA, then pipet 800 µl 3M sodium acetate pH 5.2 on the pellet and vortex vigorously. Then spin down, discard the supernatant (ALL supernatant), wash the pellet with 1 ml of 70% EtOH (you really don't want to carry over 3M NaAcetate), and then resuspend the pellet in the original amount of dd H2O.

Hi Joshua, have you looked into kapabiosystems kits? I think they have some good Tag polymerase that are resistant to the normal inhibitors .

good luck

Also I think Quanta have a brand called ToughMix that is resistant to some inhibitors.

I'm surprised that no one has mentioned using a Proteinase K based extraction. While BME and CTAB work very well at preventing oxidative stress on DNA during extraction, I have found that not having any in and doing an hour incubation in the presence of Proteinase K has given me good yield with very pure and high quality (long fragments). look at Lunde et al., 2000. HortScience 35(4):729-731. Then as others have already mentioned, skip the phenol in the last step doing either a salt based precipitation or isoamyl alcohol, just be sure to suck off any remaining alcohol instead of just air drying, that will improve the extraction also. Finally I will add that I am currently processing several hundred samples and will be doing the ProtK lysis and initial spin clean up then applying to either Bioline or BioTek Omega 96-well genomic filters. I don't lose much yield, it is adaptable to any initial lysis method, it is extremely fast and high throughput, and it only costs about 50 cents per sample. Good luck

In conclusion to this saga, this is what I have found to be the best way to deal with difficult DNA extractions from cambial tissue from Boraginaceae and Meliaceae tree samples:

DNA is extracted using the MoBio Power Plant kit (http://www.mobio.com/plant-dna-isolation/powerplant-pro-dna-isolation-kit.html) as recommended by @TreenaBurgess.

Despite samples still having some reasonable amount of contamination and/or low yields, PCR of nuclear and plastid markers (ITS1 and trnHpsbA, respectively) has been successful using the KAPA3G plant polymerase (http://www.kapabiosystems.com/products/name/kapa3g-plant-pcr-kits). After much tribulation with Taq, I thankfully stumbled across this engineered polymerase that is robust to the presence of plant inhibitors.

Would highly recommend Kapa Biosystem's KAPA3G plant polymerase to anyone with difficult plant matter due to its ability to work in the presence of inhibitors and the need of but a small amount of DNA (equal or slightly < 10ng).

Hi,

we work with tissue from trees and our experience is that in most cases the dilution of the extraction solution is sufficient.

Good luck Doris