Preparing tRNA involves a precipitation and washing process. Here's a breakdown based on the provided reference:
tRNA Preparation Protocol
This protocol outlines the steps for preparing tRNA from an aqueous solution.
Step 1: Precipitation
- To the aqueous phase containing tRNA, add 0.1 volume of 3 M NaOAc (Sodium Acetate) at pH 5.0. This helps in neutralizing the charge on the tRNA, facilitating its precipitation.
- Add 1 volume of isopropanol to precipitate the tRNA. Isopropanol lowers the dielectric constant of the solution, reducing the solubility of nucleic acids like tRNA.
- Incubate the reaction mixture at -80°C for 30 minutes. This deep-freezing step maximizes the precipitation of tRNA.
- Centrifuge the mixture at 21,000 × g for 30 minutes at 4°C. Centrifugation separates the precipitated tRNA from the solution, forming a pellet at the bottom of the tube.
Step 2: Washing
- Wash the aminoacyl-tRNA (aa-tRNA) pellet with cold 70% ethanol 2-3 times. This step removes residual salts and other impurities that may have co-precipitated with the tRNA. The cold temperature helps maintain the integrity of the tRNA.
This process results in a purified tRNA pellet ready for downstream applications. Note that the reference specifies washing an "aa-tRNA" pellet, indicating this protocol is specifically designed to purify aminoacylated tRNA. If you're starting with uncharged tRNA, it is still applicable but the resulting product will not be aminoacylated.
