HELP: RNA isolation from cell cultures

Sat Aug 5 15:57:47 EST 1995

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,

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