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.
Amino Modifiers (2071, 2141, 2149 & 2135)
No changes are required from the standard method recommended by the synthesiser manufacturer. Coupling is as per standard nucleoside amidites.
Cleavage & Deoprotection
The trifluoroacetyl (TFA) protecting group on the primary amine is removed during standard ammonium hydroxide solution deprotection. However, a minor side reaction during deprotection can lead to irreversibly capping 2-5% of the amine. This could be significant if multiple additions of the modifier are made.
To prevent the reaction, synthesise using acetyl-protected C and, prior to cleavage and deprotection, wash the column with 10-20% DEA/acetonitrile. Deprotect in AMA at 65°C for 15min.
Thiol Modifier (2191)
BTT or DCI can be used as activators. Do not use ETT. A coupling time of 10min (600s) is recommended. To facilitate AMA deprotection, dmf-G and Ac-C should be used.
Cleavage, Deprotection & Purification
Before carrying out the cleavage and deprotection, wash with a 20% DEA/MeCN solution, then:
- Cleave and deprotect using 100mM TCEP in AMA at room temperature for 2h.
- Desalt using a G25 column.
- Purify if required.
- Dry the oligo.
- Add 100μl of 87mM TCEP in water.
- Leave for 1h at room temperature.
- Pass through a G25 column pre-equilibrated with the conjugation buffer.
To conjugate, add the appropriate maleimide, acetamide or equivalent to the above solution and react as appropriate.
Once the reaction is complete, pass through a G25 column pre-equilibrated with 0.1M TEAA or, if conjugation is on a solid surface, thoroughly with water.
Storage & Stability
Store in a freezer below -10°C. Diluted samples must be freshly prepared for use and used within 24h.