Rhodamine derivatives are not sufficiently stable to survive conventional deprotection and these must be attached to amino-modified oligonucleotides using post-synthesis labelling techniques. Because Tetramethyl Rhodamine (TAMRA) is not base stable, the procedure to cleave and deprotect the labelled oligonucleotide must be carefully considered. Using the UltraMILD monomers and deprotection with potassium carbonate in methanol, TAMRA oligonucleotides can be fairly conveniently isolated. To streamline the preparation of TAMRA oligos, we offer 3'-TAMRA CPG for 3' labelling and TAMRA-dT for labelling within the sequence. We also offer TAMRA NHS ester for labelling amino-modified oligonucleotides.
Coupling: Use either dmf-dG (10-1029-xx) with standard monomers and Cap A or use UltraMILD monomers (dA: 10-1601-xx, dC: 10-1015-xx, dG: 10-1621-xx, dT: 10-1030-xx ) and UltraMILD Cap Mix A (40-4210-xx/ 40-4212-xx).
Deprotection: If standard monomers were used, deprotect in tert-butylamine/water 1:3 (v/v) for 6 hours at 60 °C. If UltraMILD monomers were used, deprotect in 0.05M Potassium Carbonate in Methanol, 4 hours at Room Temperature. See Technical Bulletin for details (http://www.glenresearch.com/Technical/TB_UltraMild_Deprotection.pdf). Note: A side reaction can occur that leads to a loss of the TAMRA label. Treatment of the support prior to cleavage/deprotection with a hindered base, such as 10% diethylamine in ACN, for 3-5 minutes will minimize the loss of label.
Freezer storage, -10 to -30°C, dry. Light sensitive material.
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%|||