|2066||PC Linker-CE Phosphoramidite|
|2122||PC 5'-Biotin-CE Phosphoramidite|
|2130||PC 5'-Amino-Modifier-CE Phosphoramidite|
|2131||PC Spacer-CE Phosphoramidite|
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.
For the PC 5’-Biotin (2122), PC Amino (2130) & PC Spacer (2131) products a 2min coupling time is recommended. For coupling efficiencies >95% with PC Linker (2066), an extended coupling time of 15min is recommended.
A second deblock step is recommended for the biotin product (2122) if the final DMTr group is to be removed on the synthesiser (the DMTr group is slow to detritylate from the N1 position of biotin).
For 2130 a column wash with 10-20% diispropylamine in acetonitrile or 10-20% DEA/acetonitrile is required prior to cleavage and deprotection of the oligo.
Both 2130 and 2131 deprotect under standard conditions. The trifluoroacetyl (TFA) group in the amino product (2130) is base labile and is therefore removed during the ammonium hydroxide or AMA deprotection leaving the 5’-amine.
For 2066, the ß-cyanoethyl group is removed under standard deprotection conditions.
Photocleavage is carried out simply by exposure of the oligo in 0.1M TEAA solution to a hand-held UV light source (~365nm) at room temperature. Quantitative cleavage occurs with a 1mW/cm2 lamp after irradiation for 10min when using products 2122, 2130 and 2131. Up to 30min with a 25mW/cm2 lamp may be necessary for 2066. The time taken for photocleavage will depend on the intensity of the lamp used. More powerful UV light sources can be used, although to avoid damaging the DNA (thymidine dimer formation) a 300nm cut-out filter is required.
When using 2066 the release of the 3’-phosphate oligo has been shown to be pH dependent. Conversion rates are higher at pH 9.4 than at 7.4. Release of the 5’-phosphate occurs directly upon photocleavage
Opaque magnetic particles are not recommended in PC-biotin-avidin capture applications. Glass particles, e.g. CPG, are best used otherwise photocleavage will be restricted to only 5-10%.
Storage & Stability
All modifiers are stored dry and protected from exposure to light in a freezer at –10 to –30°C. Stability in solution is 2-3 days. PC-modified oligos are best protected from over-exposure to light where possible.
The Photocleavable (PC) Modifiers were developed by Ambergen Inc., Massachusetts, US, Link Technologies Ltd, Bellshill, Scotland and Glen Research Corp., Virginia, US and are made available under licence from Ambergen Inc.