HELP: RNA isolation from cell cultures

Giorgio Spagnol spagnol at galactica.it
Mon Aug 21 05:48:23 EST 1995


SEGALL,LAURA,MS wrote:
> >Hi!
> >I'm working in a lab that was recently set up, and we are having 
a
> >very difficult time isolating RNA from cell cultures. We 
consistently
> >get only about half of the expected amounts, and even then, we 
seem to
> >have some DNA contamination since when we load say 5 ug RNA for 
a
> >Northern, we see a much weaker band than we do for controls, so 
some-
> >thing is interfering in our getting the proper concentration 
reading.
> >We now spin the lysed cells for 45 min. rather than 20 min, and 
there
> >seems to be some improvement, but we now think that there is 
something
> >very seriously wrong with our Guanidine Thiocyanate solution 
(although
> >we use 4M guanidine Hydrochloride with Na-citate and 10% 
Laurylsarcosyl?
> >whatever, the detergent).
> >We were told that the pH of the solution should be 7, but ours 
was at
> >5.5. We made a new solution, and got the same thing. When we 
tried to
> >fix the pH, we noticed that the citrate isn't really buffering 
the   lut
> >solution at all. We've found detergent precipitates in it and 
were
> >forced to heat it to clear it up a bit, although it didn't do 
much.
> >At this point, any help is welcome.
> >Thankyou in advance,
> >Laurie.
> 
> Dear Laurie

I know this is a simplicistic answer, but we have had very 
satisfaying results from cell cultures by using RNAZOLE!, from 
Genenco (This is the company,I think, but I could look if you 
want). We just followed manufacturer's instructions and did not 
care about pH. (A very rough approach but efficient).
Hope I've been of some help.
No affiliation etc.



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