Genomic DNA won't dissolve - will too much heat shear it? - Help! At wits end with something that should be simple (Mar/31/2006 )

Help!

I have received genomic DNA extracted by a transgenic mouse facility from mouse embryonic stem cells and cannot get it to dissolve. I am not sure what method the facility used to purify the DNA but I cannot get it to dissolve in TE. Some of the samples have a gelatinous mass that is too voscous to easily pipette and others have visible off white strands. The result is that I get an inconsistent amount of DNA in each aliquot of a sample. In an effort to get them to dissolve, I left the samples overnight at 55C which helped but did not solve the problem. Then I tried heating a few of the samples up to about 90C. This seems to have sheared the DNA (long streaky mess instead of a tight band at the top of the gel when I run it out). Is this the expected result for this high of a temperature? Also are there any otehr tricks for getting the DNA to dissolve? Thanks!

-Zdonald-

Dissolve DNA in an alkaline solution may help

The following is the DNA solubiliztion step from DNAZol protocol:

QUOTE

Dissolve DNA (without drying) in 8 mM NaOH by slowly passing the pellet through a pipette. Alternatively, dissolve DNA in water. However, the alkaline solubilization of DNA occurs faster and assures full solubilization of the DNA precipitate. Add an adequate amount of 8 mM NaOH or water to approach a DNA concentration of 0.2 - 0.3 µg/µl. Typically, add 0.2 - 0.3 ml of 8 mM NaOH or water to the DNA isolated from 107 cells or 10 - 20 mg animal tissue.

Alternatively, I found this method also helps:

QUOTE

Add DNA to 20 to 500 µl of 10 mM Tris HCl, 0.1 mM EDTA, pH 7.5. Complete dissolution of the DNA may requires several hours of agitation at 37°C or 55°C (may need overnight).

-pcrman-

Can't the concentration of DNA that u're finally getting be too much for it to solubilize completely? u have an idea as to how much it is? Can u try dissolving in a larger volume of the buffer? Or ya.. an alkaline pH would help..

G'luck!

QUOTE (Zdonald @ Apr 1 2006, 11:25 AM)

-cheeztoast-

I was going to also suggest a larger volume, an alkaline pH, and a bit of heat (37°C?). When I say an alkaline pH, I was thinking Tris buffer, pH 8.0 or so -- not 8 mM NaOH, though I don't know what the pH of this would be. I know NaOH solutions are used to denature DNA, but usually at 0.5 M or so...

The changes you saw at 90°C are likely due to the DNA strands melting apart, and coming together wrong in such a concentrated solution.

It sounds like you either have a very large amount of DNA and are trying to dissolve it in way too little volume, or your DNA needs to be further cleaned up.

What are you using the DNA for?

-HomeBrew-

I am using the DNA for Southerns to screen transfected ES cells for my recombination event so ideally I need a concentration of no less than 500ng/ul. What is the best way to further clean it up? EtOH precipitate and resuspend? Thanks
Zoe

QUOTE (HomeBrew @ Apr 1 2006, 09:02 AM)

-Zdonald-