DNA, contrary to RNA, typically does not dissolve well in acidic environment. Saliva is acidic to start with and you will carry this on with your extraction method. Then you try to dissolve the DNA in ddH2O, which is highly acidic due to the CO2 that dissolves very well in dionized water producing carbonic acid (pH 4.5). Always use a buffer to dissolve DNA, but avoid EDTA, there is no need of it and will interfere unpredictably with many reactions that might follow. Normally 20 mM Tris/HCl pH 7.4 will do. Overdrying should not be a problem, while not drying enough is a problem because the remaining EtOH will slow down re-solubilization of the DNA. This is why 37-50 °C help, since they evaporate the EtOH. Anyway solubilization of nucleic acids is an endothermic process, so heating a bit will always speed it up.

Dear Aamir Iqbal ,

Did you use absolute ethyl alcohol or isopropanol to precipitate DNA?

Hi Aamir, 

After adding ddH2O, but the tube on the shaker overnight would help. And you can use TE buffer instead of H2O to dissolve the pellet. 

I hope this help! Good luck.

Chiao-Ling Lo, TE buffer of how much concentration?

Shu Canwei, Yes I do use Isopropanol

If you dry the pellet to much it can be difficult to resuspend - particularly if you precipitated using isopropanol. 

Dear Aamir Iqbal ,

In my opinion, the pellet of DNA may be very little form your samples.

And I think you can think about these two steps:

1, after you wash the pellet with 75%  absolute ethyl alcohol, please do not make the pellet too dry;

2, incubate the pellet with ddH20 in a warmer or water bath for 5 min may help to dissolve the DNA pellet.

Good luck.

Hi Aamir, I agree don't let the pellet dry, just get rid of access 70% EtOH from your wash by re-spinning your sample and taking rest EtOH off with a Eppendorf pipette! Then incubate your pellet at 50C in a water bath for 1-2 hours with occasional vortexing. That is all I can think of in the moment. Good luck!

Is it possible that the insoluble material is just something other than DNA? If you take the resuspended sample, centrifuge it and then remove the clear supernatant, that may contain the dissolved DNA.  You can run a gel with the clear supernatant or try to use it for PCR and find out.

I agree with Dr Barker. we used to extract DNA from different plant leaves including fig and quercus, the samples didn't dissolve completely, but when checking it on the gel, and using it for PCR it was working Ok

If still not dissolved then you may use 1x TE buffer and let it overnight at 4 degree temperature

I agree with Dr. Barker and Dr Jamous. Can happen that the DNA samples do not completely dissolve. However, I usually dissolve the DNA pelet into an appropriate volume of TE 1X and to facilitate the pellet dissolve, I leave the samples at +4°C overnight.

Genomic DNA or any DNA longer than 100 kb would be difficult to be dissolved. Normally, we suspend with ddH2O, and incubate at 70C for 1 hour, with gently mix in between. You also can keep in 4C to dissolve and store there for a few months.

Hi Aamir, I think I can help. We used to work with the extraction protocol you mention, and the problems where more or less the same. So, even the price of the extraction will be higher I recommend you to purify the DNA phase after the phenol purification with microcon or amicon columns. The last step would be to add 50microL (miliQ water or TE buffer, depending on how long you'd like to keep the DNA) when you finish the washing and then flip over the top part of the device in a clean tube. Hope it works.

DNA, contrary to RNA, typically does not dissolve well in acidic environment. Saliva is acidic to start with and you will carry this on with your extraction method. Then you try to dissolve the DNA in ddH2O, which is highly acidic due to the CO2 that dissolves very well in dionized water producing carbonic acid (pH 4.5). Always use a buffer to dissolve DNA, but avoid EDTA, there is no need of it and will interfere unpredictably with many reactions that might follow. Normally 20 mM Tris/HCl pH 7.4 will do. Overdrying should not be a problem, while not drying enough is a problem because the remaining EtOH will slow down re-solubilization of the DNA. This is why 37-50 °C help, since they evaporate the EtOH. Anyway solubilization of nucleic acids is an endothermic process, so heating a bit will always speed it up.

For genomic DNA is better to let it overnight at 4oC , the pellet will dissolve and after that hold it at -20oC for further uses. 

Your problema is, as already commented, that you are drying too much the DNA to get rid of etanol. One suggestion, put your simple to dry in a thermomixer at 50°C for 5-7 min. I would be enough to eliminate etanol.

I agree with Ziguo Zhang  reply, except if you want homogeneous mix of genomic DNA you could keep the samples at 65c over night. 

Hi,

My first thought was over drying of the pellet, as Hector has suggested.

This is a common problem. As mentioned above, incubating in 65c rotating over night usually helps. Also check the ratio of amount of DNA extracted vs. amount of water you have added. You might have too much DNA which also limits the ability to dissolve completely.

As stated, you have to be careful not to over dry the pellet. Once my pellet is dissolved in H2Odd, I incubate it a 65ºC for 20min to 1 hour (as needed), and this dissolves it completely.

Dissolve DNA pellet in a given volume of TE 1X and leave the samples at 4 ° C overnight.

TE buffer or TAE buffer? Daniel Lerda

Dear Aamir,

I agree partially with Pietro, DNA will be hard to dissolve into ddH2O if not properly dried after ethanol precipitation.

I suggest that you dry completely your DNA pellet, by putting your tubes open in a 37C oven for 1 hour after ethanol removal and vaccum dry. In the meanwhile, put a ddH2O vial into a dry-block set at 70C. Dissolve your dried pellet into the corresponding volume of preheated ddH2O. If not completely dissolved, just add 5ul more of 70C ddH2O. Then check your extraction by electrophoresis in 0.8% agarose.

Have you checked your extractions?  I mean, if you run 1-2ul of your sample in agarose gel, can you see the genomic DNA band?

Some times with manual protocols involving phenol, some salts are still co-precipitated with DNA and remains un-dissolved for ever, and ever ..., so check your DNA extractions, even if not completely dissolved.

If this doesn't work, try to dissolve into LTE (5mM Tris-HCl pH 7.5, 0.01 mM EDTA pH8.0), with this very low concentration of EDTA you're protecting your DNA sample, whereas PCR, qPCR, restriction, ligation, etcc, are not compromissed!!

Hope this helps you

Tris EDTA buffer (TE buffer) will be better for dissolving DNA. And you should try giving an ethanol wash (centrifuge with 70% ethanol) to remove salts. One cannot be sure if the DNA dissolved and the precipitate you are seeing might be that of salts.

Since you are using the manual method I agree with the 70% ethanol wash, and also the EDTA in the TE should protect your DNA from enzymatic digestion. However the problem may be that you are carrying over a lot of proteins. I used the manual method for many years and thought that I was getting a lot more DNA than a colleague who used kits, but one day I found out that I didn't have more DNA but lots of proteins in my pellets. Since that day I switched to DNA purification kits, which give you cleaner pellets for further use in digestions, ligations, PCR, etc.

When the DNA pellet was not dissolve in ddH2O that means high salts and ahuge amount of proteins are the major. So, phenol cholroform prefered follwed by chloroform extraction two times and then ethanol 70. Let the pellet to dry and then dissolve it in worm TE buffer. I hope that work sith you.

1X Tris EDTA buffer and leave the samples at 4 ° C overnight

Subin Cheri Kunnumal Raj I used to do that, but recently after overnight incubation @ 4C, the pellet get swollen & not that easily to be dissolved by pipetting . Do you suggest anything to overcome swollen pellet?