Grettings.

¿Did you calculate the flux of the column with dextran blue or another compound? Maybe you lost the sample 'cause when you run the electrophoresis you don't have sample. Check that before any other and also when you purificate the DNA, use the relation OD 260/280 nm to make sure that you have DNA.

Nothing else occurs to me.

I hope that would serve.

Thanks Felipe for your prompt kind response.

I think that DNA sample was lost during the of the steps of phenol extraction or during Butanol extraction. The sephadex column we use in the lab has been used previously and tested for similar purposes. I dissolved the excised band in 150 micro L water and 100 micro L 3M NaAc pH 5.5 and the volume of the aqueous phase after phenol extraction was ~ 500. Yet the volume I could transfer of the aqueous layer was ~ 400 as I have been extremely careful to avoid agitating the phenol phase in the tube. Then I had to do 1-Butanol extraction to bring the volume to ~ 200 for better ethanol extraction with 3 times the volume of the DNA aqueous solution. I do not know which step exactly resulted in lose of the DNA.

Could you see a pellet at the time of EtOH precipitation? If so, was it white or translucent and did it dissolve easily? Was the pH of the NaAc accurate?

Try adding the NaAc after the phenol and butanol extractions, not before, so you can better control the amount of salt in the EtOH precipitation. The amount of NaAc you used is way more than you need (you had ~40% when you only need 10%).

that fact you have not a signal, results in several options: maybe you have not any more DNA or you have a problem of electrophoretic migration, your agarose maybe is so concentrated or is not completely pure, it is contaminated with other polysaccharides, salts, and proteins. these differences can affect both, the migration of the DNA and the ability of the DNA recovered from the gel and also electric field applied across the gel (gels should be run at no more than 5 V/cm)...etc

first and as suggested felipe, make sure about the presence of your DNA by measuring OD, and also eliminate any activities of RNA by using proteinase K and RNAse!

I see that you have used H2O and personally I prefer TE!

second, when you run your sample on gel, did you see a strong signal on the well or not? maybe the amount of DNA is so important and DNA remained at the well! .....

Its very frustrating to lose a DNA sample that you may have spent days working up. Tracking the quantity of DNA in each step is always a good idea. That way you will know which step the loss is occurring in. Saving spent washes and supernate from each step can help you recover DNA when it is lost. There are a few details you need to pay attention to to insure good DNA recovery during the extraction purification process. For example, when doing an ethanol precip double checking the pH or being a bit generous with the low temperature incubation time can help a lot. Using as little agarose as possible is always helpful!

There are also a few products that can help you with recovery. For example, I find that using LPA helps with my DNA recovery. You could also switch to a gel removal kit with a spin column format, Sigma makes a decent one, to aid in sample recovery.

The pH of the phenol matters. If it is acidic a substantial portion of the DNA will stay with the organic phase. Liquid phenol stock bottles as delivered are usually acidic with pH around 6 I think, but come with a small bottle of something to adjust the pH to around 8. You add the contents of that bottle to the larger phenol bottle and shake it (do that in a fume hood with double gloves on) to mix it up well. It should be appropriate then for DNA extraction. As a side note, RNA always stays with the aqueous phase and if the phenol is much more acidic, around pH 4 or something, pretty much all the DNA will stay in the organic phase leaving only RNA in the aqueous phase. The band you extracted could have been rRNA (very abundant) which was lost from an RNase step before the second extraction, if you did that.

Also, for DNA extraction you should be using chloroform and isoamyl alcohol as well, in a 25:24:1 phenol:chloroform:isoamyl ratio. Suppliers actually sell this mixture in the appropriate ratios ready to go. To my understanding the chloroform helps keep the phenol out of the aqueous phase (phenol partially dissolve into the water) and the isoamyl alcohol prevents foaming or some other annoyance - I don't recall exactly. For plant DNA extraction, folk lore recommends octanol in place of isoamyl alcohol but I don't know the purported advantages of this.

In my experiences, the phenol is not strictly necessary for most organic solvent extractions - we used to just use chloroform + isoamyl and omit the phenol as these alone are much less hazardous to spill on yourself but phenol on your skin is bad news and you won't notice immediately because it acts first as a local anesthetic. If you do spill it on yourself, rinse the area under COLD water for at least 30 minutes (seriously - 30 minutes) or it will leave a burn mark for sure.

Good luck!

When you excised the band from the gel, did you use short wave UV to visualize the band ? If so, were you careful with the length of time of exposure of your DNA band to UV? If you fragmented your DNA during excision, the pieces would be lost in the down stream treatments. Check out the article by Lynch and Pergolizzi 2010

http://legacy.jyi.org/articleimages/3659/Aug%20jyi%201.pdf

You have too many manipulations decrease those for better recovery. You failed to include your endpoint (or next step after clean up). I would do a "freeze and spin" through siliconized glass wool in a mini tube with a hole in the bottom. Of course this tube is within another tube (without a hole in the bottom) to capture your filtrate. This can be followed by a Sephadex spin column, if neccessary.

I think you lost your sample with the phenol extraction. I would used a phenol - chloroform mixture. But even so losses would occur.

Minimize your steps to increase your recovery.

One more thing following up on my previous post: a potential reason why you had the DNA after the first extraction but not the second, is the lysis buffer you used prior to organic extraction was probably pH buffered with Tris or something to pH as high as 9 or higher, and that may have been enough to neutralize an acidic phenol. But the second time, with the DNA in water, there was not anything to drive the phenol pH to 8 or above, so in the second extraction most or all of the DNA stayed with the phenol and there wasn't enough to detect on the gel. That hypothesis at least offers an explanation of why the first extraction worked but the second didn't. I agree with Michael that minimizing the number of steps will give you higher yield.

If you need long fragment, RFLP or BAC library quality DNA, stick with these organic extractions and the second purification might be worth it if you have enough input to start with. If you are going to do PCR though and shorter fragments are fine and you don't need gobs of DNA, nothing beats anion-exchange extraction and purification (Qiagen kit). You can MacGyver that with glass wool or something in place of the matrix that is in the qiagen column, but I've never done it myself.

If you want to stay on the cheap, salt-out preps are very cheap, easy, and work really well, producing high yields of DNA that will work excellent for PCR. You can even get RFLP quality DNA from a salt out prep. Salt-out may not be the best though if you have complex polysaccharides and polyphenols to get rid of, but there are things you can add to the lysis buffer that will bind them up. I would have to look those things up though.

I was also more inclined to think that the major loss of my DNA was due to the column purification particularly after I saw a big pellet forming after Ethanol 100% precipitation. Every step was a potential cause of the DNA loss for me as I was not sure as which step actually lead to the DNA loss. I did the DNA band excision under UV light in less than 3 minutes to minimize DNA damage and I pipetted as much as I could from the aqueous layer after phenol extraction but I remember that I centrifuged for 10 minutes after phenol extraction to ensure phase separation but I wonder whether DNA could precipitate in the bottom phenol layer had it been centrifuged at 14,000 RPM for 10 minutes?

I'd agree with Arthur. I used a CTAB protocol recently and using a column for cleaning DNA samples sometimes is not the best idea. Ethanol precipitation is the best approach. Eventually, if this does not work, take an unimportant sample, go through your protocol and take small aliquots of the aqueous phases in every step of the protocol and run them on a gel afterwards. This way, you know exactely in which step you lose the DNA. And, more important: in the next experiment, take backup samples ;-).

I had this problem before. I noticed that my DNA was stayed in the phenol. So, what I did was after I pipetted the aqueous layer to a new tube, I add TE buffer to the phenol layer. shake it gently to avoid breaking the DNA. Leave it for a moment until the two layers separated. Pipette the aqueous layer (TE layer) to a new tube. Then, precipitate the DNA with ethanol. I get my DNA but not as much as in the first step. Good luck !

In my opinion 14,000 rpm is too high. In my experience maximum is 13,000 rpm. Usually I centrifuge 8,000 to 10,000 to separate phenol and aqueous layer.

You might want to check your phenol. Ensure that you are using phenol buffered with Tris pH 7.5 -8; also if you have added 8-hydroxyquinoline to your phenol, check the color and make sure that it is yellow and not orange. Once your DNA is in solution, I cannot see how it would leave the aqueous phase and enter the organic phase unless the phenol is acidic. All these values of thousands of rpm mean nothing without the rotor information and time of centrifugation. Still, assuming that the values stated are 14,000 g, 13,000 g or 8,000 g, none of these can take the DNA from the aqueous to the phenol phase. The DNA will only partition to the organic phase if the phenol is acidic. It is possible for the DNA to end up in the interphase if it is associated with protein. However based on your description this is not a likely scenario.

The more the manipulation the more you lose the sample. You should therefore try to minimize too many manipulation and precipitation steps. Once you have done the phenol extraction in the beginning, the DNA should be pure enough. Why do you require a second phenol extraction? After the gel band isolation, you should consider a column purification (e.g. PrepEase) and the so called squeeze N Freeze method (BioRad), especially if you are purifying from regular agarose gels. You may try low melt agarose and agarase combination. Any other method would results in heavy loss of DNA material. I suspect that you are losing your DNA as you go through the gel melting, phenol extraction and precipitation steps. If the gel is not melted completely, a lot of DNA trapped in the agarose will get lost. If you are going to continue your current protoco, consider adding about 15-20 ug glycogen when you precipitate; and use isopropanol instead of ethanol. Glycogen is an inert materials and helps increase the mass and precipitate the DNA down.