Apoptosis - DNA ladder method

Steven L. Sabol sabol at codon.nih.gov
Fri Apr 21 16:40:43 EST 1995


In Article <3mdtqv$ils at ftp.univie.ac.at>, Christian Seelos
<christian.seelos at univie.ac.at> wrote:
>"Juergen Sasse (Path)" <js1 at mole.bio.cam.ac.uk> wrote:
>>
>> 
>> Can anybody provide an easy and reliable method for preparing
>> DNA ladders of apoptotic cells? Using my current method I have
>> problems with the genomic DNA of control cells because the high
>> molecular-weight DNA tends to crawl out of the gel slots and
>> gives smears. On the other hand, purifying away soluble fragments
>> from the HMW DNA leaves blank control lanes (because there is low
>> apoptosis background in my control cells) which is not really 
>> satisfying.

Well-prepared genomic DNA purified by the usual SDS/PK/RNase methods is of
such high molecular weight that it is difficult to suspend to homogeneity,
to pipette, to load onto a gel, to determine concentration by OD, etc.  One
solution is to break the DNA up a little by pipetting the solutions up and
down a fixed and constant number of times in a Pipetman pipettor.  The
amount of such trituration should be the minimum required to suspend the DNA
properly.  It helps to warm the DNA solutions to 50 degrees before
triturating and before any pipetting to reduce the viscosity. The
concentration of the DNA solution that you apply to the gel should be as low
as possible to minimize artefacts due to a highly viscous sample.  

In my experience the trituration step(s) I recommend to suspend the DNA
evenly does not result in significant smearing, visible by EtBr
fluorescence, of degraded DNA on a regular (non-pulsed field) agarose gel,
so it does not interfere with detection of an apoptotic DNA ladder unless
the ladder is so faint that it requires detection methods more sensitive
than EtBr staining.

Steven Sabol
Laboratory of Biochemical Genetics
NHLBI
National Institutes of Health
Bethesda, MD 



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