|0251||TAMRA NHS Ester in DMSO|
|2434||3'-TAMRA CPG S|
|2435||3'-TAMRA CPG L|
Physical & Dilution Data
Dilution volumes (in ml) are for 0.1M solutions in 10% THF in anhydrous acetonitrile. Adjust accordingly for other concentrations. For µmol pack sizes, products should be diluted as 100µmol/ml to achieve 0.1M, regardless of molecular weight.
TAMRA-labelled oligonucleotides require milder deprotection conditions: use tbutylamine/methanol/water (1:1:2).1 Therefore, use Ac-dC and dmf-dG when synthesising the oligo being labelled. Standard ammonium hydroxide deprotection significantly degrades TAMRA. Post-synthetic labelling using TAMRA NHS Ester (0251) is discussed below.
TAMRA-dT-CE Phosphoramidite (2143) is dissolved in 10% THF in anhydrous acetonitrile (4059) to standard concentrations. Allow 15min for the product to completely dissolve.
2143 – A 6min coupling time is recommended.
Cleavage & Deprotection
2143 – Deprotection is carried out using tbutylamine/methanol/water (1:1:2) for 2.5h at 70˚C.
2434/5 – Typical protocol is as follows:
- After the oligo has been synthesised, remove the support from the column and add 600μl of tbutylamine/methanol/water (1:1:2) solution.
- Heat at 70˚C for 2.5h..
- Pass the sample down a G25 column equilibrated with water. This removes the deprotection solution.
- Sample is now ready for purification.
Poly-Pak™ Cartridge Purification2
Note that the TAMRA label degrades during deprotection and these degraded oligos are not removed during Poly-Pak™ purification.
Typical protocol is as follows:
- Connect a syringe to the female luer of the Poly-Pak™ II cartridge and terminate the male luer in a waste vessel.
- Flush the cartridge with 4ml acetonitrile followed by 4ml 2M TEAA.
- Following synthesis, deprotect the oligo as outlined above.
- Add 1 volume 0.1M TEAA with 8% acetonitrile followed by 8 volumes deionised water to the cartridge.
- Load the sample solution onto the cartridge. Reload if necessary after addition of a few drops of 2M TEAA which increases the affinity of the oligo for the support.
- Flush the cartridge with 4ml 2% TFA; do this whether the oligo is DMT ON or OFF.
- Rinse the cartridge with 10ml 1:3 – 1:4 solution of acetonitrile:2% TFA. Note: the percentage of acetonitrile used depends upon the length and sequence of the oligo. Using a 1:4 ratio is appropriate for oligos approximately 20 nucleotides long.
- Flush the cartridge with 4ml distilled water.
- Rinse the cartridge with 10ml of 0.1M TEAA containing 12% acetonitrile. This is to remove non-labelled oligos. If significant TAMRA is seen coming off the cartridge, stop the rinse.
- Elute the product with 2ml of 50% acetonitrile in water.
Post-Synthesis Labelling Protocol
As noted above, TAMRA-labelled oligos will not survive standard deprotection conditions. Therefore, if such conditions are necessary, 0251 can be used to label the oligonucleotide post-synthetically. This is provided as a 0.17M solution in DMSO. A synthesised amino-modified oligonucleotide is then conjugated to TAMRA using the NHS ester in sodium carbonate/bicarbonate buffer (0.1M, pH=9). A typical protocol for the conjugation of an amino-modified oligo (synthesised at 0.2μmol scale) is as follows:
- Dissolve the amino-modified oligo in 125μl of conjugation buffer.
- Add 6μl of TAMRA/DMSO solution (ca. 5-fold excess).
- Vortex the mixture and incubate at 37°C in the dark overnight.
- Separate the oligo-TAMRA conjugate from salts and free TAMRA by size-exclusion chromatography on a NAP-10 column or equivalent.
- To do this, equilibrate the NAP column with 0.1M TEAA.
- Load the reaction mixture onto the column and let this flow into the column.
- Add 0.5ml TEAA buffer and also let this flow into the column.
- Elute the conjugate with <1.5ml TEAA buffer.
- Collect the conjugate and reduce to a suitable volume for HPLC purification.
Storage & Stability
All products are stored dry in a freezer at –10 to –30°C, protected from light. Labelled oligos are stored in the dark. 2143 is stable in solution for 24h.
- Automated synthesis of double dye-labelled oligonucleotides using tetramethylrhodamine (TAMRA) solid supports, B. Mullah and A. Andrus, Tetrahedron Lett., 38, 5751-5754, 1997.
- Poly-Pak™ Purification of 3’-TAMRA Labelled Oligos, v1.0, 5/99, Glen Research. Poly-Pak™ is a trademark of Glen Research.