Cleavage and Deprotection of Unmodified Trityl-On DNA Oligos

Materials

  • 5 ml Luer-slip syringes (Becton Dickinson #301028) - supplied with oligos
  • 1 dram glass vials (Wheaton #224702) and green Teflon-lined caps (All-Pak #5200) - supplied with oligos
  • • Concentrated ammonium hydroxide (28-30%), ammonium hydroxide (Baker #9721-01)
  • If oligos are to be dried:

Note: It is critical to use fresh ammonium hydroxide to ensure complete cleavage and deprotection. We recommend using small 500ml bottles stored tightly capped at -20°C and discarded if they have not been used up within a month.

Cleavage

  1. Draw 1.5mls of ammonium hydroxide up into a 5cc Luer-slip syringe; remove any trapped air.
  2. Firmly attach the syringe to the bottom of the synthesis column.
  3. Secure a second 5cc syringe to the top of the column, pushing the syringe bodies (not the plungers!) together firmly to ensure that they will not fall apart later.
  4. Slowly push approximately 300 uL of ammonium hydroxide from the lower syringe into the upper syringe. It may be helpful to gently pull up the plunger on the top syringe while pushing the ammonium hydroxide from the bottom syringe.
  5. Allow the column to incubate with ammonium hydroxide for 15 minutes at room temperature. (It may be helpful to place the syringe-column assembly 
in a 1000 ml beaker to keep it upright during the incubation).
  6. Repeat steps 4 and 5 three more times until the column has incubated for 1 hour in the ammonium hydroxide.
  7. Push the remainder of the ammonium hydroxide from the lower syringe into 
the upper syringe.
  8. Invert the assembly, and manipulate the syringes so as to get all of the liquid into what 
is now the lower syringe (if necessary, you can add a little air by fully depressing the top plunger, removing the top syringe while gently pulling down on the lower plunger, and then replacing the top syringe). These contortions are necessary because ammonium hydroxide has a tendency to drip out of the syringe if the tip is down.
  9. While keeping the lower syringe pointed upwards, remove the top syringe and the column. Invert a glass vial over the tip of the lower syringe, and smoothly invert both together. Push down on the syringe plunger to dispense the ammonium hydroxide solution into the vial. Cap the vial tightly using the green, teflon-lined cap. (Avoid over-tightening the cap since this may cause the neck of the vial to break - note that the caps have crushable foam under the teflon and hence are not re-usable).
  10. With a Sharpie marker, draw a line on the vial to indicate the bottom of the meniscus. This will serve as an indicator to assure that the vial remained sealed during the deprotection.

Deprotection

  1. Incubate the sealed vial for 1 hour at 65°C.
  2. Following the deprotection, chill the vial for 10 minutes at -20°C. 
(Caution: Do not attempt to open the heated vial until it has been thoroughly cooled!!).
  3. After chilling, check the ammonia level to verify that the vial remained sealed and that the ammonium hydroxide concentration remained constant. If the level is significantly below the mark, the oligo should be transferred to a fresh vial, dried, resuspended in ammonium hydroxide and re-deprotected (use a fresh cap!) 
to ensure complete removal of the base-protecting groups.
  4. The oligo can now be purified via a Glen-Pak Cartridge. If the oligo needs to be dried, we recommend gentle evaporation under a stream of air or inert gas in order to preserve the 5’ DMT.