HELP: RNA isolation from
harold.drabkin at channel1.com
Sat Aug 19 01:42:00 EST 1995
Subject: HELP: RNA isolation from
To: BK5U000 at MUSICB.MCGILL.CA
Subject: HELP: RNA isolation from cell cultures
B>We were told that the pH of the solution should be 7, but ours was at
B>5.5. We made a new solution, and got the same thing. When we tried to
B>fix the pH, we noticed that the citrate isn't really buffering the
lut B>solution at all. We've found detergent precipitates in it and were
B>forced to heat it to clear it up a bit, although it didn't do much.
B>At this point, any help is welcome. B>Thankyou in advance, B>Laurie.
I routinely isolate total RNA from COS1 and CV1 cells; the original
reason is to get acylated tRNAs to analyze by northern blots for %
acylated vs deacylated. The standard protocols to do this use a pH 5
buffer for the isolation to preserve the acyl linkage. However, the
yields were pathetic with what I knew we could get using a pH 7 buffer.
We did a systematic study of yield vs pH, and found that yield dropped
precipitously as the pH was lowered below 6.5.
We weren't using the guanidine buffer, but a buffer designed originally
for pH 7 work to keep nuclei intact, so that they could be pelleted.
(the reason being that precursor tRNA might migrate at about the same
position as acylated-tRNA (which runs slower than unacylated tRNA in the
acid-urea gels used for the final analysis)). I was amazed that the pH
had such an effect, since the detergent was NP40. The buffer does have
some EDTA in it.
When the cells were viewed under the microscope during lysis, it was
seen that the lower pH's needed much longer to lysis (as judged by the
clearing of cell-boundaries on the TC plate).
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