I think your isolated gDNA have EtOH due to un-proper drying. So you have to reprecipitate the gDNA and drying again.

Thank you Raghvendra. I will re-precipitate the DNA again.

I had the same problem when trying to set up yeast DNA extraction. I tried re-precipitating, as well as a number of other things (even purification with other protocols). Finally I solved it by performing a final clean-up passing the sample through a sephadex G-50 column. Alternatively I believe you can pass the sample through a filter for DNA (eg. Pall).

Thanks Cristina, I have had limited success using spin columns. I washed my gDNA samples by placing them in a spin column, then adding the standard kit Pre-wash buffer, then wash buffer, and finally the elution. It worked for some, but others it didn't seem to help at all.

I think I will try the sephadex G-50 columns after I have re-extracted DNA from tissue (luckily I have lots of muscle tissue to work with) using more phenol:chloroform:Isoamyl-alcohol (25:24:1), and chloroform:isoamyl-alcohol (24:1) washes and to make sure I get rid of all ETOH during the drying of the gDNA pellet.

I initially used SDS in my extraction buffer for my samples, and I have read that this may inhibit PCR if it is present in my DNA. I will remove this from the protocol as well to see if it helps.

I use SDS as well, and I also tried spin-column based kits, with limited success. My results were similar to yours: it worked for some, it did not for others, and I could not determine why. I suspend sephadex G-50 in H2O (even though I've read that may be problematic for some sephadex, I've not had any problem). I clean up a lot of samples, so I do it in 96-well plates that have a 30-40 um filter at the bottom (any filter with a large enough pore size will do), but you can also prepare the columns with plastic pasteur pipettes or any other way. I suspend Sephadex G-50 in ddH2O (leave at the very least 2 h to hydrate, best overnight), place 400 ul of the suspension (shaking before pipetting), and compact the column by centrifuging at 750 g for 90 sec. Discard the H2O collected, place your sample on the column (NO MORE than 50 ul for this height of column), and centrifuge again at 750 g 90 sec. The liquid that comes out of the column this time should be your clean DNA.

To remove phenol contaminant you should to wash twice with cloroform before preciptation. To avoid salt contaminants try to preciptate only with isopropanol.

I would suggest to do an isopropanol precipitation (1:1) followed by a wash in 70% EtOH. It is essential that there is no EtOH left. What we normally do is add 1ml of 70% EtOH, spin, discard supernatant, spin again, remove as much as you can with a pipette and let it dry for 10 min at room temperature.

Alejandro, do you mean you can add ether to the sample right after the step of phenol chloroform? I mean:

1.- phenol-chloroform

2.- diethyl ether

3.- EtOH and precipitate

or should it be:

1.- phenol-chloroform

2.- EtOH and precipitate

3.- diethyl ether

4.- EtOH precipitation again?

I never tried this one. I'll check it out next time, see if I can skip sephadex. So far, without sephadex, some of the samples of any given batch work with PCR and some other samples do not work, but I'll see with your modification if all of the samples work.

HI, THe best way to purify genomic DNA, especially if you have limited starting concentration and do not want to loose is to use a ultrafiltartion unit. This is the best: Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-30 membrane.

Good luck!

The best way to clean up phenol extracted DNA is to perform a chloroform extraction followed by dialyzing against TE buffer. If you have a small amount of DNA you can form a punch a hole in a small tube, cover with dialysis membrane, and close lid. The smaller the surface area the more time you need to dialyze.

I recommend you don't precipitate the DNA with alcohol as you'll irreversibly bind some of the leftover proteins to the DNA. This is a problem only if you extract with phenol.

Wow thanks everyone! I have a lot to process here, so I'll read up more about each of your recommendations and hopefully it works!

Hi Brent, one final note :) If it´s a probleme of too much salt maybe it is useful to dialyse your probe. nice day, norbert

it may be possible there are some salt contamination. u should used 70% ethanol wash and u can use glycerol, BSA or DTT in PCR reaction mixture to avoid the other contamination in PCR reaction mixture that inhibit the PCR ampliphication

If you have enough DNA you can try a quick and dirty experiment by doing a serial dilution of your template and compare the dilutions.

If this and the methods suggested above do not work you might try to purify the DNA with a commercial Kit.

Also, what's the GC content of your DNA? Usually DNA with lots of GC is tougher to melt, and you need particular conditions in the PCR. I know this from a friend having problems with it, and for that purpose they use DMSO or betaine.

The GC content of my amplicons are about 47.8% average... I have tried dilutions of 1:9 (DNA extract:water) and then a further dilution of that to a 1:99. None of these worked.

I have tried extra phenol washes, chloroform washes, and further cleaning with a zymogen gDNA extraction kit (of which about 60% worked, but still not good enough).

I recently tried a Nucleospin kit, which worked on four samples... I'm just slightly apprehensive to invest in a full kit after only testing four samples (I could only get hold of a few from a fellow student).

Is that also applicable to plants?