|
Epicentre Forum 1 (2)
Recovery and purification of RNA from in vitro transcription
reactions can be tedious, time-consuming and result in significant product
loss. For example, one of the more common methods involves digestion
of the DNA template using RNase-free DNase I, followed by gel filtration
to remove unincorporated nucleotides, and then phenol extraction to remove
proteins and ethanol precipitation to concentrate the RNA. A second standard
method is purification of the RNA by polyacrylamide gel electrophoresis,
then extraction of the RNA from the gel by electroelution.
Table 1 presents an easier and faster method. Ammonium
acetate selectively precipitates only RNA, leaving DNA, proteins, and
unincorporated nucleotides in the supernatant. Recovery of RNA transcripts
longer than 100 bases approaches 100%.
Table 1. Selective RNA Precipitation
with Ammonium Acetate
- Add ammonium acetate solution to the transcription reaction
to a final concentration of 2.5 M. (e.g. add one volume of 5
M ammonium acetate and mix.)
- Chill mixture on ice for 15 min.
- Centrifuge in a microfuge at high speed (approx. 10,000 x g)
for 15 min.
- Remove supernatant and wash pellet with 70% ethanol to remove
salts.
- Resuspend the RNA pellet in the desired volume of RNase-free
H2O or buffer.
- Quantitate RNA by an appropriate method.
- Note: Since ammonium acetate decomposes by loss of ammonia,
solutions should be prepared only from the pure salt which has
been kept cool in a closed container. Sterilize only by filtration;
do not autoclave. Store solutions at +4°C.
|
|
