my dna won't label!

Karen Ferri kferri at email.unc.edu
Tue May 20 17:35:22 EST 1997



Hi all!

I have prepared DNA to be used as a probe in the following manner:

Digested plasmid with appropriate enzymes, then ran the digest on a gel.
Excised the insert and purified on a Millipore column, followed by
phenol/chlorform extraction.

The OD260/280 ratio was pretty good; 1.79.  Ran a mini gel to confirm DNA
was there and it clearly was recovered.  The size is ~ 2.5 kb.

I have prepared 2 insert preps b/c I just can't get it to label with p32.
We use random priming protocol and even after overnight incubation,
nothing.  Labeled the control DNA of the kit and that labeled great, so
it's not the random priming kit.

Which means it is my DNA but I don't understand why.  It should be very
"clean" and the OD is good.  I have labeled other dna for probing that had
worse od's and slightly smaller in size (2.1 kb) and labeling worked.  

I even added 5 min. to our protocol for the denaturation step before
adding the kit reagents and still nothing.

Any idea?  I am under pressure b/c my boss needs this so I can probe some
blots that she is using as the basis for a paper.

TIA

Karen



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