Richard T. Pon
The University of Calgary
Synthetic oligonucleotides must be treated with concentrated ammonium hydroxide as part of the post-synthesis work-up to remove protecting groups from phosphate linkages and exocyclic amino groups. The strong odor of ammonium hydroxide makes working with the solutions difficult and so many core facilities evaporate or lyophilize these solutions before distributing them. However, this evaporation can be time-consuming and the ammonia vapor is very corrosive.
This article describes how our laboratory modified a Savant SVC100H Speed-Vac to obtain fast evaporation using an inexpensive water aspirator (Fisher Scientific Airejector #09-956 or #09-958). A water aspirator is a better vacuum source than a mechanical oil pump to evaporate ammonium hydroxide solutions, because ammonia vapors damage most vacuum pumps. Ammonia will still slowly corrode the aspirator, but it is much easier and cheaper to replace an aspirator every six months (about $15) than it is to overhaul a vacuum pump (about $700).
Water aspirators produce a weaker vacuum, limited by the vapor pressure of water, than oil-based pumps so evaporation takes longer. Fortunately, evaporation can be accelerated simply by increasing the operating temperature of the evaporator. Savant Speed-Vacs have built-in heaters. Newer models have several heater settings, but older models can be heated only to a single temperature, about 37 degrees. We found that both the Savant internal heater and the external Savant accessory heater, which shines a halogen lamp through the Plexiglas lid, did not give us fast enough evaporation. We also found the external heater unsatisfactory because the bright lamp could damage fluorescently labeled oligonucleotides by photobleaching.
While it may have been possible to adjust or replace the internal thermostats (there are two connected in series) to obtain a higher temperature, we found it more convenient to simply disconnect the heating element from the internal controls and to control it instead with a variable transformer (0-120 V, 10 A, Staco Energy Products, Fisher Scientific #09521-110). Adjustment of the heater's voltage allows crude but sufficient temperature control. Setting the variable transformer to about 55 V produces a surface temperature around 50-55 degrees. This is about the same temperature used to deprotect oligonucleotides overnight and so should not damage the DNA. The temperature can be monitored by a stick-on liquid crystal thermometer (Bio-Rad #165-3720), but we simply check the temperature by touch. Even higher operating temperatures might be possible but we have not raised the temperature further because of concerns that this might damage the evaporator's Plexiglas lid.
The following is a step-by-step procedure for modifying a Savant SVC100H Speed-Vac, a model with a single heater setting. Models with multiple heater settings probably do not require this modification.
1. Disconnect the evaporator, turn it upside-down, and remove the bottom cover (six screws).
2. Locate the heating element. It is a rigid 1/4 inch diameter black tube located in a small recess around the circumference of the evaporator's bowl.
3. Disconnect the heating element by slipping off the female spade connectors from each end. Cover the ends of the two disconnected wires with insulating electrical tape to prevent any shock hazard. They can be easily reattached if the heater's built-in thermostats need to be restored.
4. Prepare a line cord with a three-pronged male 120V plug by cutting the female end off a common AC appliance cord. Separate and strip the ends of the three exposed wires and attach a female spade connector to each of the black and white wires and a fork connector to the ground (green) wire. Use a crimping tool to attach these connectors.
5. Pass the end of the cord through the grill in the bottom cover and tie a knot in the cord to keep it from being accidentally pulled out. Attach the white and black wires to the male spade connectors on the ends of the heating element; the element does not have "polarity" so the wires can be attached in any orientation. Loosen one of the hex head screws from the bottom of the evaporator bowl, slip the ground (green) wire under it, and tighten.
6. Reattach the bottom cover.
The evaporator will now have two line cords, the original one for the motor and a new one for the heater.
The evaporator is most easily connected to the water aspirator by using two pieces of rubber vacuum tubing that are joined in the middle by a plastic quick-connect fitting (Bel-Art). The quick-connect fitting eliminates the need for a valve and also allows larger diameter tubing (from the evaporator) to be attached to smaller diameter tubing (to the aspirator). When using a water aspirator, it is essential to always release the vacuum (i.e., disconnect the hose) before turning off the flow of water. Otherwise, water may back into and flood the evaporator.
The Speed-Vac RH24-18 rotor we use can concentrate as many as 24 screw-capped glass vials (15 x 45 mm) at a time. We make two to three dozen oligonucleotides per day so one modified evaporator can accommodate our daily throughput. Our oligonucleotides are usually diluted in about 2 ml of ammonium hydroxide. Concentration of these samples to about 0.75 ml takes only an hour when the evaporator is heated to 50-60 degrees. The concentrated, ammonia-free solutions are then desalted by direct application to prepacked NAP-25 Sephadex columns (Pharmacia). By eluting these columns directly into microcentrifuge tubes, we are able to concentrate and desalt twenty-four oligonucleotides in only a couple hours. We have used the modified heater for almost ten years now and our maintenance costs for vacuum pump repairs have decreased dramatically.
The author may be contacted at The University of Calgary, University Core DNA Services, Dept. of Medical Biochemistry, Calgary AB T2N 4N1, Canada.
Return to the The ABRF Home Page