alkaline lysis -- unrealistic spec readings

Ed Siefker via methods%40net.bio.net (by ebs15242 from creighton.edu)
Wed Nov 19 16:54:10 EST 2008


We ran out of miniprep columns, so I'm trying out the alkaline lysis
protocol in Maniatis (CSH).  When checking the DNA on my spec, I get
a nice shaped curve, with good ratios, but the concentration is
unrealisticly high. I'm getting readings of around 2 ug/ul.  When I run
it on a gel and actually visualize the DNA, it can't be more than
200ng/ul tops.

I can't figure this out.  My spec still reads known DNA concentrations
correctly. There aren't any smears or anything that would indicate genomic
DNA or RNA contamination.  This DNA digests just fine with EcoRI too.

Anyone have any idea what's going on here?  I need to have these plasmids
sequenced, but I'm not sure what to tell them the concentration is.  Thanks
-Ed



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