quandries on Genomic DNA purification - troubleshooting (Apr/13/2002 )
What is a crystaline precipitate that I get sometimes (not always) when I precipitate my genomic DNA preps and how can I get rid of it? My theories are protein(that was not completely removed during extraction), salt(I use 3.5M KOAc pH 5.2) or SDS(form the extraction buffer). Any thoughts would be much appreciated.
frofsa,
The stuff that is precipitating in your sample may be a lot of things. What method are you using (phenol, salt method or kit of some type)? I would guess that it is protein precipitating out with your DNA. Usually if the isolated DNA does not go into solution when you add TE or water, this indicates that there is protein. You may have to re-isolate your sample if this is the case.
AronD,
I am using the old fashioned phenol/chloroform extraction method. I use an extraction buffer that has 10mM Tris/0.1M EDTA/ 0.5% SDS. The stuff that precipitates will go back into solution upon addition of water or TE so I guess I could rule out protein. If it is a salt coming from a component of the extraction buffer,what would be a relatively easy way to remove it from my sample. I have found that it is not really soluble in 70% EtOH.
frofsa,
Hmmm, sounds like what you are describing is not salt contamination, otherwise it should be removed by the 70% ethanol. It could be SDS or something else. You may want to try salt/ethanol re-precipitation of these samples and see if you can get the the DNA to precipitate out without the white stuff. Otherwise you may have to go back and do a second phenol extraction (yuck!). You may also want to run the genomic DNA on a gel to see what it looks like or take 260/280 readings on a UV spec to check for protein or other contaminants. Good luck!
The stuff is most likely EDTA, which is insoluble in 70% ETOH above pH 6.0 or so. We have seen this many times using 0.1 M EDTA. Sometimes your KOAc drops the pH sufficiently and sometimes it does not. Remember that EDTA binds 4 mol of monovalent cation per mol. The excess EDTA dissolves in TE or water but will inhibit Mg++ dependent enzyme activities. Solutions: 1) use less EDTA in your lysis buffer (10X TE buffer, pH 8.0 is convenient), 2) use NaOAc pH 5.2 (the Na salt of EDTA seems to be more soluble). 3) rinse an EDTA contaminated DNA precipitate with 70% ETOH containing 0.2 M NaOAc, pH 5.2. Do not expose to low pH for too long to avoid depurinating the DNA.
(Edited by drstressor at 3:04 pm on April 16, 2001)
(Edited by drstressor at 3:04 pm on April 16, 2001)
drstressor,
I checked all of my reagents used in the extraction buffer individually and it turned out that it was the EDTA that was precipitating out, especially when ice cold ethanol was added as opposed to room temp EtOH. I had never heard of anybody having a problem with this before. Now I use a final concentration of 0.02M EDTA in my extraction buffer (which I think will be enough to inhibit DNases) and I haven't had any problems. I cleaned up the EDTA contaminated samples by diluting and reprecipitating them with as little room temp ethanol as possible and this seemed to prevent the crystalization. Thanks for the valuable information.