DNA lost dramatically during storage - (Sep/20/2012 )

Hi, I stored genomic DNA extracted from human saliva in 10mM Tris(pH8.0) at -20C for<1month. Today I thaw it, and test concentration again by nanodrop, and find that the concentration drop more than half (eg. drop from ~500ng/ul to ~200ng/ul).

I wonder what cause it drop so fast? May it be endonuclease contamination? If so, I think the cleaved DNA should still show absorb at 260nm.

May it because DNA attached to tubes? I use polypropylene tubes.

Please give me some suggestions. Thanks a lot!

try mixing or vortexing and read absorbance again. maybe it precipitated. what is your blank?

I'd strongly recommend storing your DNA in TE rather than in Tris buffer.

Curtis on Thu Sep 20 22:39:55 2012 said:

Thanks! I blanked with Tris, which I used to resolve DNA.

As my samples are genomic DNA, will it get sheared if I vortex it? I did mixed with finger flip though.

phage434 on Fri Sep 21 00:44:34 2012 said:

I read sometines EDTA interfere with PCR assay. I wonder if it is safe to use TE for qPCR? Thanks!

Yes, in sufficiently large amounts, it will chelate the magnesium from your buffers. But usually the fraction of a PCR reaction that is template DNA is 1-2%, so the effect of 1 mM EDTA in the template DNA sample on the 1-2 mM magnesium concentration of a typical PCR buffer is tiny. It's much more important that the DNA is really still there.