|2134||5'-Fluorescein-CE Phosphoramidite (6-FAM)|
|2136||5'-Hexachloro-Fluorescein-CE Phosphoramidite (HEX)|
|2137||5'-Tetrachloro-Fluorescein-CE Phosphoramidite (TET)|
Physical & Dilution Data
Dilution volumes (in ml) are for 0.1M solutions in dry acetonitrile (4050). Adjust accordingly for other concentrations. For µmol pack sizes, products should be diluted as 100µmol/ml to achieve 0.1M, regardless of molecular weight.
6-FAM (2134), HEX (2136) & TET (2137) – A 3min coupling time is recommended.
2148 – A 12-15min coupling time is recommended.
2139 – A 15min coupling time is recommended.
2068 – A 10min coupling time is recommended.
All CPG supports are used in a manner identical to standard nucleoside supports.
Non-nucleosidic CPG supports do not detritylate as rapidly as nucleosidic ones, therefore an additional detritylation step is recommended. It is therefore necessary to use a cycle that does not contain an initial capping step.
2134, 2137, 2148 & 2139 can be deprotected following standard protocols with ammonium hydroxide solution, although 2137 should not be subjected to prolonged heating at 55°C.
2136 is also deprotected with ammonium hydroxide solution, but at RT for 24h. The ammonia must be removed immediately after deprotection. Do not use tbutylamine/methanol/water (1:1:2) as this will completely degrade the HEX.
Oligos containing 2068 can be deprotected with ammonium hydroxide using standard conditions, removing the fluorescein protecting groups at the same time.
2366, 2359 & 2368 – Cleavage of the oligonucleotide from these supports requires 45min at room temperature with ammonium hydroxide. Complete the deprotection using the protocol required by the nucleobases.
2370 – Deprotect using protocols required by the nucleobases.
Fluorescently labelled oligonucleotides can be purified and analysed using the same methods employed for standard DNA. Note, however, DMT ON purifications are not possible with 2134, 2136 or 2137 as these are 5’-modifications with no DMTr group. Nevertheless, the hydrophobic nature of these modifications make purification straightforward.
It is also possible to carry out analysis on dilute solutions using fluorescence detection with RP-HPLC (see below for data).
Storage & Stability
All phosphoramidites and CPGs are stored dry, frozen at –10°C to –30°C. 2134 and 2137 are stable in solution for 1-2 days and after 4 days show <90% coupling efficiency. 2139 is stable in solution for 2-3 days, 2068 & 2136 only for 24h, and 2148 is unstable and must be used immediately after preparation.
Fluorescently labelled oligonucleotides must be stored in the dark, either dry or in neutral aqueous media at –20°C. Do not store crude fluorescently labelled oligonucleotides in ammonium hydroxide solution.
Fluorescein Labelling of RNA
It is possible to label an RNA oligo at the 3’- or 5’-end with fluorescein and deprotect/desilylate leaving the label intact, provided the desilylation is carried out using Et3N.3HF in DMSO (1:1) in place of TBAF.