Monday, October 20, 2003

Description
This protocol describes the desilylation or 2' deprotection of synthetic RNA.

Procedure
1. After synthesis on the 1 M scale, take the column apart with pliers and deposit the resin in a glass deprotection vial (see Hint #1).

2. Add 4 ml 3:1 NH4OH:Ethanol and incubate at 55C for 5 hr.

3. Take a sample of the liquid and determine the absorbance at 260 nm so you can calculate the yield from the synthesis. Measure the A260 of a 1:40 dilution of the sample (for the 1 M scale). The absorbance equals the A260 multiplied by the dilution factor multiplied by the volume (ml) of the preparation.

4. Dry down the preparation without heating in amber 2 ml microcentrifuge tubes.

5. Dissolve the sample in 10 l/A260 Units of Triethylamine Trihydrofluoride. Mix by vortexing and stir for 24 hours in a 2 ml amber microcentrifuge tube.

6. Quench the reaction with 2 l/A260 Units of ddH2O.

7. Add 100 l/A260 Units of 1-Butanol.

8. Incubate overnight at -20C.

9. Centrifuge for 20 min at high speed in a microcentrifuge and dry in a speed vacuum concentrator.

10.Dissolve the RNA in 10 l/A260 Units in ddH2O. The RNA is now ready for purification (see Hint #2).


Recipes
3:1 NH4OH:Ethanol 30% (v/v) NH4OH: 100% Ethanol


30% (v/v) NH4OH


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