alkaline lysis -- unrealistic spec readings

Dr. Hiranya S. Roychowdhury via (by hroychow from
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.
> Thanks
> -Ed
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Hiranya S. Roychowdhury, Ph.D.
Asst. Professor,
Health & Public Services
Dona Ana Community College
New Mexico State University
Las Cruces, NM 88003

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