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MOLECULAR BIOLOGY: WORKING WITH PROTEINS
PROTEIN EXPRESSION: In Vitro Translation
PREPARING RNA FROM RETICULOCYTE LYSATE
Preparing RNA from Reticulocyte Lysate
Contributor: The Laboratory of Andrew Murray at Harvard University Overview
This protocol requires Reticulocyte Lysate as a starting material. See the protocol on the Preparation of Reticulocyte Lysate for instructions to obtain this material. Procedure
1. To 1 ml of lysate add 330 μl of 0.1 M Acetic Acid (see Hint #2).
2. Pellet the precipitate for 5 min in a microcentrifuge at full speed.
3. Discard the red supernatant.
4. Suspend the pellet in 100 μl of NETS.
5. Add an equal volume of Water-Saturated Phenol and mix thoroughly.
6. Centrifuge for 5 min at full speed in a microcentrifuge at 20°C to separate the phases.
7. Recover the aqueous phase and repeat the Phenol extraction (see Hint #3).
8. Add 2.5 volumes of 96% Ethanol to the aqueous phase and mix well (see Hint #4).
9. Centrifuge for 30 min at full speed in a microcentrifuge at 4°C.
10. Remove as much of the supernatant as possible and rinse the pellet in 80% or 96% Ethanol, re-centrifuge, completely remove the supernatant again and allow the pellet to air dry.
11. Dissolve the pellet in 50 μl of ddH2O or 1 mM EDTA.
12. Check the concentration of the RNA sample by UV absorbance (see Hint #5).
13. Store the RNA sample at -70°C or lower.
1 mM EDTA 80% (v/v) Ethanol 96% (v/v) Ethanol NETS 1% (w/v) SDS
200 mM NaCl
50 mM Tris, pH 8.0
1 mM EDTA
0.1 M Acetic Acid Water-Saturated Phenol (CAUTION! see Hint #1) See protocol for the Preparation of Water-Saturated Phenol. BioReagents and Chemicals
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. The Acetic Acid lowers the pH to between 5 and 5.5 which precipitates ribosomes, tRNA and many of the components of the protein synthesis machinery. This is often known as the pH 5 precipitate.
3. Back extract the Phenol phases with NETS if desired and combine the washings.
4. It is critical to mix the sample well after the addition of Ethanol.
5. One mg/ml of RNA reads 23 at 260 nm. However, many other substances absorb at 260 nm, particularly Phenol.