I have encountered the similar problem as you. I have isolated chicken
blood genomic DNA by using phenol/chloroform method but I suffered from the
high viscosity of the DNA. I would like to use the DNA for fingerprinting
(i.e. restriction digestion and gel electrophoresis).
I think there are two problems to cause the high viscosity, firstly is the
existence of carbohydrate in the DNA since carbohydrate cannot be removed by
phenol/chloroform extraction. In this case, how carbohydrate can be
removed? Secondly, TE or water is too little to dissolve the DNA well (i.e.
the concentration of DNA is too high). In this case, we can use much more
TE or water to dissolve the DNA. But the question is what is the suitable
DNA concentration to avoid the high viscosity.
Thanks a lot
Wilson
"Gayatri Mani" <sweechie at HOTMAIL.COM> wrote in message
news:19990712044939.55323.qmail at hotmail.com...
> To whom it may concern,
> I had some trouble isolating genomic DNA from CHO cells.The DNa I
extracted
> was very very viscous and even after restriction enzyme digestion,the DNA
> was still quick viscous and when I tried to load the wells,they popped
> out.Can anyone suggest why this may be so?
> I extracted the DNA via a phenol/chloroform extraction protocol.I had a
> doubt about the quality of the phenol-choloform used,hence I have now
> resorted to a new bottle.Could this be the problem?
> Any suggestions would be appreciated.
> Also, I was wondering about the washing conditions for a southern blot.I
was
> using stringent washing conditions however was told to try a less
stringent
> method.My first wash is with 2xSSC (very briefly) then for 15minutes (x2)
of
> 2xSSC/0.1%SDS at Room temp and then a 30 minute wash with 0.2xSSC/0.1%SDS
at
> RT.My signals are rather weak and only are barely distinguishable at about
> 15 days exposure at -80degrees.Any suggestions would be appreciated.
> Thanks.
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