5 replies to this topic

[joy123]

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!

[Curtis]

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

[phage434]

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

[joy123]

Curtis, on 20 September 2012 - 02:39 PM, said:

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

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.

[joy123]

phage434, on 20 September 2012 - 04:44 PM, said:

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

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

[phage434]

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.