OU’s 454 reagent cooling system To extend the life of the GS20 reagents for longer than 42 flows, we found it necessary to develop an apparatus for reagent cooling. Implementing this cooling system has allow us to obtain highly accurate reads for control and sample DNA beads past 230 bases. This cooling system is composed of a 1/4" copper coil in an ice chest, a 3/16" copper tubing in the reagent tray, and a water pump all connected by 1/4" Tygon tubing. The ice chest is half filled with crushed ice plus one liter of water. The 3/16" copper tubing is routed around the large reagent bottles and then around the small reagent in the shape of an "M". After the bottles are placed in the reagent tray, the tubing is pushed down to the top of the separating ridges in the sunken bottle holders. Chilled water is then poured in the gap around the bottles until it covers the copper tubing.

In our home-made cooling system, composed of a 1/4" copper coil in an ice chest connected to 3/16" copper tubing in the reagent tray and a water pump by 1/4" Tygon tubing, water in the closed pumping system is chilled as it is pumped through the 1/4" copper coil in the ice chest. The water then flows to the 3/16" copper tubing in the reagent tray. After picking up heat from the reagents the water flows back to the pump where it starts another cycle. The 0.1amp AC cooling pump located in a plastic tray is placed just behind the Styrofoam ice chest

Top view of the reagent bottle holder with the 3/16” copper cooling tube The styrofoam ice chest containing the 1/4” copper cooling coil before and after adding ice & water to cover the cooling coil

Description of the waste removal component of the system To keep the waste water from heating up the reagents, we remove the waste from the sequencer during the run. This is accomplished by diverting the flow from the larger blue tube that is the drain for the primary pumps and the two smaller tubes that are the drains for the purge pumps to a 20 liter plastic bottle placed beside the Roche/454GS20 sequencer. Thus, one end of a 1/4" Tygon tube is placed in a 20 liter waste bottle beside the GS20 sequencer and the other end is inserted through the back vent of the sequencer behind the chiller compartment and connected to a 1/2" to 3/8" plastic elbow. This tube comes up in the back left corner of the reagent compartment of the sequencer - in the gap between the compartment and the rounded corner of the reagent tray and this reagent tray must be all the way back in the compartment, touching the back wall. The Tygon tube then was run horizontally across the back of the tray over to the waste opening in the back right corner of the reagent tray and attached to the 1/2" to 3/8" plastic elbow that collects the outflow from the 3 bundled, overflow sipper tubes. As the sipper assembly is lowered on to the reagent tray, the elbow is aligned with the three waste tubes on the assembly. The elbow rests in the 3cm gap between the waste opening in the tray and the bottom of the sipper assembly. The three waste tubes now drip into the elbow instead of reagent tray. The length of the tubing inside the sequencer must be carefully adjusted so that the tubing does not bow upward between the elbow and the back left corner of the compartment. Also, the waste liquid dripping into the elbow must have a downhill path out the back of the sequencer and down into the waste bottle. This system is shown on the next slide.

Drain tube diverter to re-route discarded liquid reagents to an external capture bottle Diverter and overflow tube placed so that the waste reagents flow to an external capture bottle instead of the hollow, white plastic bottle holder.

Finally an Aluminum plate is placed over the Sipper to prevent it and the reagent bottles from rising up when the bottles are surrounded by chilled water, the lid is closed as best it can be and the run is begun.