Hi Kelly,
are you sure that this is not RNA that you see at the bottom of the  
gel? RNA is much more abundant and you may have ignored the fainter  
band much further up.
wolfgang


> Hi Kelly,
>
> Are you trying to do PCR or a Southern with your DNA?
>
> Dan Dickinson
> Stanford University
>
> Date: Fri, 8 Jan 2010 01:01:38 -0500
> From: [log in to unmask]
> Subject: [DICTY - 253] Degraded DNA after cell lysis
> To: [log in to unmask]
>
>
>
>
>
>
>
>
> I am trying to clone a deletion strain of Dicty using homologous
> recombination.  I am currently using a lysis buffer
> which contains 500 uL of .5 M KCl, 50 uL of 1 M Tris (pH 8.3), 250 uL of 50
> mM MgCl2, 22 uL of 100% NP40 and 22 uL of 100% Tween 20 in a 5 mL
> solution.  I am mixing this lysis buffer with Pro K (1 uL for every 25 uL
> of Lysis buffer).  Then I am adding cells in log phase and heating at 95
> degrees for 10 min. in a thick walled ependorff tube to deactivate the Pro
> K.  I have tried many ranges of cell culture to lysis buffer (1:1, 1:2,
> etc.), but when I run the DNA conformation gel, my bands keep  
> appearing at the
> bottom of the gel.  It appears that the DNA is being degraded.  I
> don't know why this is happening.  Does anyone have any thoughts?
>
> Kelly
> Dunning
> Graduate Assistant
> University of Central Arkansas
> Biology
> Department
> 003 C Lewis Science
> Center
>
>
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Wolfgang Nellen
Abt. Genetik
Universität Kassel
Heinrich-Plett-Str. 40
Germany
phone: ++49 (0)561 804 4805


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