hroychow at NMSU.EDU
Wed Nov 6 16:49:19 EST 1996
At 06:16 PM 11/5/96 -0600, Juan Pablo Martinez-Soriano wrote:
>At 13:21 11/5/96, Dr. Hiranya Sankar Roychowdhury wrote:
>> The way I store my RNA sample is as follows:
>> I estimate the concentration of the RNA spectrophotometrically and then
>> store the standard RNA solution in 66.66% ethanol (ie. 2 vols) at -70 C.
>66.66% ethanol, then the rest is water?
>If this is the case, RNA will be in solution and exposed to any contaminant
>RNAse you would have in the water.
If RNase is present, it will destroy the RNA even in its ppt. form over
time. While preparing and storing RNA, we assume that everything coming in
contact with the sample is free of RNase.
>The rationale of using salts is
>precisely that.. to take the RNA (or DNA) out of solution and making it
>"inert" to any nuclease action. Am I correct or did mess it badly?
You are partially correct. But, while DNase activity may be mostly arrested
while frozen, RNase activity has been known to go on (albeit at a vv slow
rate) even at -20 C. The time it takes to thaw a sample tube of RNA from
-70 to 0 C is sufficient to allow significant RNA loss if there is RNase
already present in the sample. Theoretically, one should be able to store an
RNase-free sample of RNA in buffered solution at room temperature.
>> each use, I aliquote the required amount and add either NaCl or NaOAc to ppt
>> the RNA for 15 min at -70 C.=20
>We take OD readings to estimate concentration, then precipitate with salted
>ethanol (95%) and store at -20 or -70 C. When needed, we mix well the tube,
>take an aliquote of an estimated amount, and proceed to spin and resuspend.
Each to his own device.
Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu
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