Depending on the amount of organic compounds extract once or twice with phenol (saturated with TE, pH 7.5-8.0). The water phase (is on top) volume, should be +/-equal to the organic phase volume. After that you can extract with an equal volume of Phenol/TE-Chloroform-Isoamylalkohol (50:48:2). Optiional an extraction with an equal volume of Chloroform-Isoamylalkohol (24:1). After that an extrakt three times with Diethylether (!! caution, the Dieethylether is here the upper phase). Incubate 10 min at 65°C and precipitate your DNA.

Try phenol-chloroform extraction (1:1). Mixing the sample very well with this clean my DNA. It works. Good luck

Dear Joshua

In a desperate situation you can also try a n-butanol extraction. This is specially useful if you get phenol contamination after a phenolic extracion.

Good luck

Thank you both for your answers!

I have never worked with plants but I think you could try to mix 1:1 v/v with ether to clean residual phenol or other organic compounds. Normally you will know from the absorption spectrum if it is phenol.

Freeze and if you don't want to wait, .pipette out the ether which is liquid Repeat a couple of times. it is very quick if you put your samples in the -20 or -80 freezer.

Check the Ab260 Ab280 or perform a scan. If all that fail than use a kit to clean your DNA.

@Abdelhalim, could you clarify for me: when you say 1:1 v/v do you mean 1 volume of DNA extraction solution TO 1 volume ether?

Thanks!

You can try to clean are using chloroform: isoamilic alcohol (24:1). It can substitute the phenol:chloroform solution . Besides this you can add PVP (0.009g/ sample) and beta-mercaptoethanol (2%) in your extraction buffer (CTAB for example)

Hi Joshua! Yes

Phenol is efficient at cleaning DNA, but remnants will disturb further enzymatic reactions, even after alcohol precipitation. Therefore a phenol (pure, saturated with TE, pH adjusted), then a phenol-chloroform should be followed by some solvent removing the phenol. Chloroform does it better than alcohols, so doing a pure chloroform extraction after any phenol mix is good to do before e.g. ethanol precipitation. Note that these extractions require the two liquid interphases to be in good contact with each other, but the more they mix ( -> emulsion) the higher is the risk of shearing the DNA. If you need long genomc DNA (>5kb, estimation) it is not good to shake too much during the extraction. Old methods... Maniatis...

Hi Jushua! I meant you mix 1 volume of your disolved DNA in TE buffer with 1 volume of ether. Ether is just to clean residual organic compounds left after the extraction. Sorry, I didn't mean extraction solution. Perform a UV scan.

well, have you centrifuge the sample twice when you add the washing buffer or the phenol?

Actually I am not familiar with CTAB technique, because I am working with stool and using kit.

but, maybe you can try it once :)

hope it works

Joshua, try adding PVP in your CTAB buffer during grinding and you can use PCI(phenol:chloroform:isoamylalcohol) in equal vol followed by a Chloroform:isoamyl alcohol (24:1) to remove all your phenolic compounds. I have used this method on wide variety of plants which have high phenolic content as well as resins...worked for me...gudluck

A simple protocol that just works:

http://www.ncbi.nlm.nih.gov/pubmed/19146820

Hi Joshua, it sounds like you have quite a lot fo clean up to do. Since you were extracting from plant material, one of the possibilities for high 230nm absorbance could be the failure to remove the carbohydrate load. I have a tough way and an easy way for you.

The tough way is to go all the way back to the Ctab step since that is the step for carbohydrate removal, and start from there. I don't know the exact proportion to mix but I am sure there are protocols out there for clean up of DNA prep using Ctab. PCI afterwards should take care of the protein load. After this full blast clean up protocol, you should be able to retrieve clean DNA.

The lazy way you can try is to use DNA extraction kit columns which comes with cleaning reagents. Most of these column will allow you to load DNA prep and perform washing on the column and then elution of the clean DNA. Do take note of the maximum load each column can take though. For elution, you can usually elute twice to minimise loss and concentrate down later if you need.

All the best!

in the cleaning step add 1M tris 200micrl and pvp(0.1%) 10micrl + 2% beta mercaptoethenol 100microl

One of the major problem with DNA extraction is the coextraction of water soluble contaminants. If these contaminants are free (not bound to DNA) you can simple get rid of them by using ultrafiltration washing. I use amicon filters of 100 kDa or 30 kDa, with very good results. If the impurity is bound to the DNA you must first break this bound. You can do this chemically (e.g. playing with pH. Alkaline pH strips DNA from its electrostatic charges and thus its molecular interactions.Playing with hydrophobic or hydrophylic solutions) or physically (e.g. playing with electric separation, micro-electroelution, microelectrodialyse or non linear electrophoresis).

Depending on the amount of organic compounds extract once or twice with phenol (saturated with TE, pH 7.5-8.0). The water phase (is on top) volume, should be +/-equal to the organic phase volume. After that you can extract with an equal volume of Phenol/TE-Chloroform-Isoamylalkohol (50:48:2). Optiional an extraction with an equal volume of Chloroform-Isoamylalkohol (24:1). After that an extrakt three times with Diethylether (!! caution, the Dieethylether is here the upper phase). Incubate 10 min at 65°C and precipitate your DNA.

Hello ..As far as for my best of my knowledge and i overcome this problem with Phenol-Chloroform method for extraction of DNA.Please find attached protocol for your use..All the best.

see the protocol for DNA extraction from Vainio and Hantula, 2000 from wood total DNA isolation. They use Phenol:clorophorm:isoamyl alcohol and later clorophorm:isoamyl alcohol.

good luck with it!

if you have 500ul DNA, add 2*500ul cold absolute Alcohol, then centrifuged at 12000 rpm for 5 min , discarded the supernatant allow the DNA precipitate to dry and then added 200 ul TE buffer.

Add 1ml of 70% ice cold ethanol to your DNA and centrifuge and repeat two to three times and dissolve it in DNAse free water or TE buffer

I agree if you do not want to start again with the purifying you have to include a phenol/Chloroform extraction to clean up your DNA. It also helps to make a Proteinase K digestion before (you mentioned that also the 280 ratio is high, which means you have still some proteins in the solution). I just recently isolated Norway spruce DNA from needles with the CTAB method (Chang) and the 260/230 ratio was 0.2-1.3. After Proteinase K (Neb3 buffer+ 20ng/ml ProtK 37°C 1 hours) digestion and phenol/chloroform extraction (repeated as many times till I do not have interphase) I get 1.97-2.02 230/260 ration. I can suggest using PeqGold Phase Trap A columns to avoid picking up some interphase. After that you have to precipitate your DNA in the presence of salt. If you have high amount of DNA use only 1.5-2.0 times volume of EtOH and try to coin up the DNA on a glass goad. It is important to wash the pellet very carefully with 70% of RT alcohol to remove the salts. Be sure that the pellet is swimming in the wash solution so, that also from under the pellet you can remove the salt. To dissolve the DNA use low concentration of Tris buffer (5mM pH:8.0) but without EDTA. EDTA has also absorption at 230nm.

I have reasons to believe that DNA is more stable than it is generally considered to be. The DNA, once extracted and contaminated or not, is amenable to analyses with electrophoresis, PCR, restriction enzymes etc. Now, it depends on what you do with the ‘contaminated’ DNA that you have extracted using CTAB method.

Do not worry about contamination if sufficient amount of DNA is available. Decontaminate the DNA only when your experiments demand precise quantities of high quality DNA.

Try your analyses with the ‘contaminated’ DNA that you have in stock. I am sure that the DNA – contaminated as you report – shall fruitfully yield to further analyses. Kindly inform me about either success or failure in this regard despite my being sure of success.

Use Phenol-Chloroform extraction especially 2 ml buffered Phenol and 2 ML chloroform:Isoamyl alcohol (24:1).

If your DNA is suspended in a buffer or H20 try adding 1/10 volume of 3N Sodium Acetate(mix). Heat at 65 degrees 10 mins. Centrifuge for 5 mins at ~10k rpm. Place supernatant in fresh new tube and add 1 volume of cold IsoPropanol. If possible "loop" out your DNA and wash in 80% ethanol. Dry DNA and resuspend

How to get rid of protein contamination if it persists even after phenol chloroform extraction?