Coupling: 3 minute coupling time recommended. The use of UltraMILD monomers are preferred. (Catalog Numbers: dA: 10-1601-xx, dC: 10-1015-xx, dG: 10-1621-xx, dT: 10-1030-xx). To avoid any exchange of the iPr-Pac group on the dG with acetyl, use the UltraMild Cap Mix A (40-4210-xx/ 40-4212-xx). 0.02M Iodine for oxidation is recommended.
Deprotection: If UltraMILD reagents were used, deprotect in 0.05M Potassium Carbonate in Methanol for 4 hours at Room Temperature OR for 2 hours at Room Temperature in 30% Ammonium Hydroxide. If standard bases were used, deprotection in Ammonium Hydroxide at Room Temperature for 24-36 hours will give acceptable yields. However, the oligonucleotide will require a reverse phase purification, e.g., Poly-Pak™ or Glen-Pak™ purification.
QUESTION: Does AMA or methylamine cause any degradation to fluorescein or fluorescein-type dyes such as FAM or FITC?
RESPONSE:Response: While AMA (Ammonium hydroxide/40% Methylamine 1:1 v/v) is considered compatible with fluorescein, the use of methylamine when deprotecting a Fluorescein-labeled oligo does lead to a small amount of degradation, which is characterized by a the appearance of a late-eluting peak by RP HPLC that shows no visible fluorescein absorbance. With standard deprotection conditions (AMA 10 minutes at 65 C) the amount of degradation is approximately 5%
The table below shows pack size data and, for solutions, dilutions and approximate couplings based on normal priming procedures. Please link for more detailed usage information with the various synthesizers.