EtBr and heat denatured DNA

Alan KAY kay at albert.lyon151.inserm.fr
Thu Feb 26 17:15:55 EST 1998


In article <34F32991.67BB at ipk-gatersleben.de>, boernke at ipk-gatersleben.de 
says...
>
>Hi all,
>
>lately I prepared carrier DNA for transforming yeast using the Gietz
>protocol. I sonicated the solution as long as the average fragment size
>ranges around 7 kb which was judged from an agarose gel. I than heat
>denatured the DNA and quickly cooled on ice in order to keep it in a
>single stranded manner and subsequently checked it again on a gel. But
>this time all the DNA, except for some low MW smear, seemed to have 
>disappeared. Does EtBr not properly stain ssDNA or did I something wrong
>in principle?
>
>Cheers
>Ricky 
>
>******************************************************************
>
>Frederik Boernke
>Research Group of  Molecular Plant Physiologie
>Institute for Plant Genetics and Crop Plant Research (IPK)
>Corrensstr. 3
>06466 Gatersleben
>Tel.  039482 -5 321
>Fax. 039482 -5 515
>e-mail: boernke at ipk-gatersleben.de
>http://www.ipk-gatersleben.de

Hi,

several of the replies to your question have some part of the truth. EtBr 
is an intercalator, between the bases of nucleic acids. The fluorescence 
observed in visible light during UV illumination is due to energy transfer 
from the base to the intercalated EtBr molecule, which then emits the 
visible light (or it may be the other way around, I can't remember, but 
since nucleic acids absorb at about 260 nM, the first is most probable). In 
double stranded nucleic acids, the bases are oriented on basically a 
horizontal plane, because of the hydrogen bonding between the bases of both 
strands. This is what is called stacking, because on the same strand, 
adjacent bases will stack one on top of the other (and the same is true of 
the bases on the other strand). EtBr will then intercalate between two 
bases, on the same strand, and will itself be stacked. Energy transfer 
between the bases and the EtBr molecules is favoured by this stacking,which 
depends on the correct alignment of EtBr with the bases, resulting in an 
intense fluorescence. In single stranded nucleic acid molecules, the bases 
are not stacked (except in regions of strong secondary structure), and are 
oriented more or less randomly. Only those EtBr molecules which manage to 
stack efficiently between two bases will be able to emit fluorescence. As a 
rule of thumb (that means guesswork), to see a denatured DNA size marker 
(for example in a glyoxalated RNA gel) I use 5 times as much DNA as I would 
in a normal gel where the DNA size marker is double stranded. I hope this 
helps.  




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