Using Minor Bases on an Expedite Synthesizer

The Expedite 8900 series synthesizers are excellent DNA synthesizers, which use very small amounts of amidites per coupling cycle. However, the volume of reagent needed to purge, dry and prime the lines and valve block can require multiple priming steps when changing an amidite. This is a significant disadvantage, especially when using an expensive amidite. There are, however, a few measures that can be taken to decrease the volume used by the Expedite 8900 series synthesizer.

• Amidite Dilution. The Expedite synthesizer operates at an amidite concentration of 20ml per gram. This is equivalent to a 58-67mM solution depending on the base. Therefore, 100µmole of an amidite should be diluted in 1.5 ml (50µmole in 0.75ml) yielding an amidite concentration of 67mM.
• Line Filter. The top half of the line filter in the minor base vial can be easily cut back to 1cm with a razor blade. This will minimize the amount of amidite lost in the frit. Do not cut the bottom portion of the line filter.
• V-vial. Another option is to use a 5ml V-vial, available from Wheaton (cat. # 986279). When using these vials the tubing may need to be curled (around a pencil or other tube) so that it fits in the vial. Also, the end of the frit may be trimmed to a V shape (this must be done carefully so as not to expose the tubing) or removed altogether. If the frit is removed the amidite solution must be filtered to ensure that it does not have any particulates.
• Priming Protocol. Even more critical is the protocol that the operator uses to prime the monomer. Perseptive Biosystems does recommend multiple priming of the minor bases through both columns to dry and fill the lines. Their recommended protocol, however, unnecessarily utilizes expensive monomer to achieve what good anhydrous acetonitrile (ACN) can accomplish. The following is an alternate priming protocol designed to minimize amidite loss during priming.

1. Put 5ml of anhydrous acetonitrile in a clean dry amidite vial and put this vial on the synthesizer in the minor base position that is to be used.
2. In the "Tools"-"Diagnostic"-"Fluids" mode, using column #1, flush lines with the ACN, first using the "flow" command, then using the volume command. Repeat this process for column #2. This will wash the lines and valve block with anhydrous acetonitrile and help scavenge any moisture present in the lines.
3. Repeat as necessary until the vial is empty.
4. Repeat the "flow" command 3-5 times to flush ACN from the frit and lines. This will dry out the lines with helium so that the amidite loaded will not be diluted.
5. Remove the empty vial and wipe the minor base line and frit with a Kimwipe.
6. Put the vial containing the amidite to be used on the synthesizer in the minor base position and prime using either the "Prime All" or "Prime Monomers" command. These prime procedures use 10 pulses to prime each of the amidites. This will consume approximately 160µl of amidite. If you have excess amidite you might want to repeat the "Prime Monomers" to prime with an additional 160µl of amidite. Note the "Prime Individual" command uses 20 pulses (320µl) to prime each reagent selected. Experienced users can use the "prime individual" and stop the cycle after counting 10 pulses.

Amidite Consumption:

Maximum number of couplings per pack (Approximate depending on actual flow rates)