alkaline lysis -- unrealistic spec readings
Dr. Hiranya S. Roychowdhury
(by hroychow from nmsu.edu)
Thu Nov 20 10:23:43 EST 2008
It has been my experience that alkaline lyses do give high yield and conc.
The yield is usually better than that from a column. I am not sure why
you are not seeing the conentration on a gel.
> 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.
> Methods mailing list
> Methods from net.bio.net
Hiranya S. Roychowdhury, Ph.D.
Health & Public Services
Dona Ana Community College
New Mexico State University
Las Cruces, NM 88003
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