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Using Thiol-Modified Oligos

Two types of reagents can be used to introduce a thiol group at the 5' end of an oligonucleotide: the 5'-Thiol-Modifier-C6 and the 5'-Thiol Modifier C6 S-S (Disulfide).

Modified oligos terminated with the 5'-Thiol-Modifier C6 are shipped with the trityl protecting group still attached to the sulfur. In order to free the thiol group for use, remove the trityl group and reduce the sulfur.

Oligonucleotides terminated with the 5'-Thiol-Modifier C6 SS (Disulfide) will need to be reduced before use.

A single 3'-Thiol Modifier is available, the Thiol Modifier C3 S-S (Disulfide). Oligonucleotides with the 3'-Thiol Modifier C3 S-S (Disulfide) will need to be reduced before use.

Procedure for 5'-Thiol-Modifer C6 Oligonucleotides
(5'-Trityl-S-(CH2)6-phosphodiester bond-oligonucleotide-3')
1. Resuspend the lyophilized oligonucleotide in 0.1 M triethylammonium acetate (TEAA), pH 6.5 at a concentration of approximately 100 A260 units/milliliter.
2. Add 0.15 volumes of 1M aqueous silver nitrate solution, mix thoroughly, and leave to react at room temperature for 30 minutes.
3. Add 0.20 volumes of 1M aqueous DTT (dithiothreitol) solution, mix thoroughly, and leave at room temperature for 5 minutes.
4. Centrifuge the suspension to remove the silver-DTT complex. Remove and keep the supernatant. Wash the precipitate with 1 volume of 0.1 M TEAA. Centrifuge and combine the supernatant with the first volume. (Alternatively, vortex the suspension and apply to desalting column equilibrated with the desired conjugation buffer).
5. Remove the excess DTT from the supernatant by gel filtration and proceed directly to the conjugation reaction. (If this free thiol oligonucleotide is not to be used immediately, it should be stored under an inert atmosphere to avoid oxidative dimerization to the disulfide.)

Procedure for 5'-Thiol-Modifier C6 SS and 3'-Thiol-Modifier C3 SS Oligonucleotides
(5'-(CH2)6-S-S-(CH2)6-phosphodiester Bond-Oligonucleotide-3') or
(5'-Oligonucleotide-phosphodiester bond-(CH2)3-S-S-(CH2)3-OH-3')
1. Resuspend the lyophilized oligonucleotide in TE, pH 8.3 - 8.5 at a concentration of approximately 100 A260 units/milliliter.
2. Reduce the disulfide bond by treatment with 0.1 M DTT (dithiothreitol) at room temperature for 30 minutes.
3. Remove the excess DTT from the supernatant by gel filtration and proceed directly to the conjugation reaction. (If this free thiol oligonucleotide is not to be used immediately, it should be stored under an inert atmosphere to avoid oxidative dimerization to the disulfide.)

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