HELP: RNA isolation from cell cultures
spagnol at galactica.it
Mon Aug 21 05:48:23 EST 1995
> >I'm working in a lab that was recently set up, and we are having
> >very difficult time isolating RNA from cell cultures. We
> >get only about half of the expected amounts, and even then, we
> >have some DNA contamination since when we load say 5 ug RNA for
> >Northern, we see a much weaker band than we do for controls, so
> >thing is interfering in our getting the proper concentration
> >We now spin the lysed cells for 45 min. rather than 20 min, and
> >seems to be some improvement, but we now think that there is
> >very seriously wrong with our Guanidine Thiocyanate solution
> >we use 4M guanidine Hydrochloride with Na-citate and 10%
> >whatever, the detergent).
> >We were told that the pH of the solution should be 7, but ours
> >5.5. We made a new solution, and got the same thing. When we
> >fix the pH, we noticed that the citrate isn't really buffering
> >solution at all. We've found detergent precipitates in it and
> >forced to heat it to clear it up a bit, although it didn't do
> >At this point, any help is welcome.
> >Thankyou in advance,
> 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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