Manual Phosphoramidite Addition

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Sometimes, perhaps to minimise the use of an expensive modifier, you may wish to perform a manual addition of a phosphoramidite in oligonucleotide synthesis. A protocol for this using a ABI 394 DNA/RNA synthesiser is detailed below (but can be adapted for use with other synthesisers).

  1. Synthesise the oligo up to the point of the addition of the modification (DMT OFF).
  2. Take up 250μl of activator in a syringe and pass this through the column.
  3. Place the column back on the line that attaches to the top of the synthesis column on one of the column positions on the synthesiser (do not attach the bottom of the column to the synthesiser).
  4. Carry out a reverse flush (on an ABI 394: Manual, Function 2 and choose the column position you need to use).
  5. Remove the column from the synthesiser.
  6. Take up 150μl of the amidite in a syringe and load this onto the column containing the part-synthesised oligo.
  7. Take up 150μl of the activator and load this onto the other end of the column such that the two reagents (amidite and activator) mix on the column.
  8. Pass the reagent backwards and forwards through the column with the syringes for around 1min.
  9. Leave the coupling reaction to stand for the desired coupling time occasionally passing the reagents between the syringes.
  10. Remove the reagent from the column and place on one of the column positions on the synthesiser.
  11. Wash the excess reagents from the column with acetonitrile (on an ABI 394: Manual, Function 42 and choose the column position you need to use).
  12. Carry out a reverse flush as per step 4.

You can now either carry out the oxidation and capping steps between syringes with washing and drying steps to remove the excess reagents in between or you can run a “synthesis” (e.g. 5T) where there is a connector (union) in the column position and acetonitrile in the “amidite” position (on an ABI 394: bottle position 5) and swap the connector for the column just after the coupling step so that all other steps are carried out on the synthesiser.

If the modification is at an internal position within the oligo then the remainder of the sequence must now be completed.

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