Materials & Reagents

Materials/Reagents/Equipment	Vendor	Stock Number
Disposables
384 well plates	Nunc	TX62409-074L
96 well plates	Falcon	358075
96 well plates	Nunc	62408946
Stock Solutions
Bleach	KIK International	7930-53325
deionized water	N/A	N/A
Reagents
Equipment
Prep Trak	LBL	N/A
100 UL Needles	Robbins	1029-44-1

Procedure

Start Up:
1. Release emergency stop button: Locate yellow and red emergency stop switch on top/front of Prep Trak cabinet and release stop button by turning knob as arrows indicate.

2. Turn on Computer: Locate tan push button switch box on top of Prep Trak cabinet, and push button labeled Computer.

3. Turn on Prep Trak: Locate black box marked Prep Track Main power, next to computer switch box, and switch on.

4. Turn on Main Air switch: Locate Main Air switch behind the Prep Trak. Place in the UP (on) position.

5. Fill the De-ionized Water Supply Reservoir: (see sub-operating procedure).

6. Turn on Vacuum Pump: Open doors to reservoir bottles and locate switch on floor. Place in the I (on) position.

7. Turn on Liquids Air Pressure: Locate Liquids Air Pressure switch inside cabinet, next to Vacuum Pump. Place in the UP (on Position).

8.Make sure that the Bleach Concentration Selection Valve is open, high or low as needed.

9. Fill Bleach Supply Reservoir: IMPORTANT SAFETY ISSUE: See sub-procedure.

10. Close Bleach Vent Valve: Located next to the Bleach Fill Valve.

11. Turn on Bleach Air Pressure Switch: Locate Bleach Air Pressure Switch under Bleach Vent Valve.

12. Load destination plates: Place plates in the cassettes as dictated by procedure for current protocol being used (see figures 3, 4, and 5). Position on fetcher and stacker platforms carefully. If the cassettes are not seated level on their platform, the tongue of the elevator will catch on a plate and potentially knock the cassette off the platform.

13.Turn on Belt Switch: Locate push button Belt Switch, under Computer Switch.

14. Start "Prep Trak" program: If program is not already up, locate Prep Trak icon on the computer desktop and double click the left mouse button.

15. Start Computer Protocol: Follow procedure for running protocols.

Shut Down:
1. Turn off belts: Turn off Belt Switch, when last plate has transferred to a cassette.

2. Open Bleach Vent Valve.

3. Turn off Bleach Air Pressure Switch.

4. Empty Effluent (waste). See Sub-Procedure.

5. Turn off Vacuum Pump Switch: Located on floor with Reservoir tanks.

6. Turn off Liquids Air Pressure switch: Located next to Vacuum Pump Switch.

7. Turn off Main Air Pressure switch: Located in back of Prep Track I.

8. Turn off Prep Track Power Switch: Locate black box in top of Prep Track, next to computer.

9. Exit out of program.

10. Turn off Computer: Locate push button Computer switch on tan box above Belts Switch.

11. Set Emergency Shut Off button.

Reagent/Stock Preparation

The bleach concentration that we use now is 15%. We DO NOT use Clorox bleach because that brand seems to be stronger than the brand we normally use.

1. We use Pure Bright: full strength bleach.

2. Pour 750 ml of bleach into glass measuring flask.

3. Pour into designated plastic jug.

4. Fill jug with de-ionized water up to black line~ 5 Liters

Prep Track Robot I (Manual) Sub-Procedures

Summary:
This collection of procedures gives information on how to fill the water reservoir manually, how to fill and drain the high concentration bleach reservoir manually, how to drain the waste reservoir manually, and how to understand and use the different computer protocols.

Procedure for filling the water reservoir manually: See figure 2.
1. Pull out reservoir tray.

2. Turn off Vacuum pump and Liquid Air Pressure in cabinet. Wait for air pressure to release, then open lid to water reservoir.

3. Roll cart with full water tank next to Prep Track.

4. Take clip off end of tube and place end of tube in Water Reservoir.

5. Unscrew lid off water tank and open it’s valve.

6. Watch water fill until it reaches the line on the water reservoir.

7. Close valve and lid on water tank, place clip on tube, and roll back out of the way.

8. Put lid back on the Water Reservoir.

9. Slide reservoir tray back into cabinet while pressing green button located on side of railing.

10. Turn Liquid Air Pressure and Vacuum Pump back on.

Procedure for Filling the High concentration bleach reservoir manually See figure 1.
WARNING: ALWAYS WEAR SAFETY GLASSES!

1. Important: Turn off the Bleach Air pressure Switch and open the Bleach Vent Valve before starting to fill. (if you do not do this the bleach will shoot up at you when you open the fill valve and lid).

2. Open the Bleach Fill valve underneath funnel.

3. Pour bleach mixture in slowly, as to not overflow the funnel, until bleach reaches the line marked on bleach reservoir.

4. Close the large Bleach Valve and the Bleach Vent Valve.

5. Turn on Bleach Air Pressure to resume running.

Procedure for draining the High Concentration bleach reservoir manually: See figure 1.
Normally, we drain any left over bleach on Friday, after draining the effluent, because the bleach concentration gets too strong after a couple of days.

1. Make sure that the Main Air Pressure Switch to Prep Trak is still turned on.

2. Pressurize the Bleach reservoir by closing the Bleach Vent Valve and Bleach Fill Valve, and turning on the Bleach Air Pressure Switch.

3. Make sure that the Effluent transfer container is in place with tube securely placed inside, and valve on transfer container is in OFF mode.

4. At the Bleach reservoir under the Prep Track, turn the red Drain Valve to the open position.

5. Close the red Drain Valve when the reservoir is empty.

Procedure for draining the Effluent (waste) reservoir manually:See figure 2.

1. Turn off the Bleach Air Pressure Switch and close the Bleach Vent Valve.

2. Make sure transfer container is in place and it’s valve is turned off.

3. Pull out the reservoir tray under the Prep Track and turn the Effluent Valves on the tray panel to their Drain positions.

4. When air is observed in the clear Drain Line, the reservoirs are drained.

5. Close the Effluent valves by turning them to the Operate position.

Figure 1. Bleach vent and Air Pressure control panel

Figure 2. Effluent (waste) control panel

On Prep Trak I, the protocols we use are:

Devel
MT384 F mix R1R2 pro.
MT 384 N mix R1 R2 pro
Redoes

Devel- (transfers from a Falcon 96 well plate to another Falcon 96 well plate).
-Inoculate 1. SU pro( which is the Set Up for the procedure)
--Inoculate I.pro (Procedure)

MT384 transfer (transfers from a Nunc 384 well source plate) -Falcon (destination is to a Falcon 96 well plate)
--Mix (aspirates twice in the source plate)
---MT384 F mix R1R2 SU.pro ( Set Up procedure)
----MT384 F mix R1R2 pro. (procedure)

Prep Trak I is programed to do redos. The procedure is as follows:

MT 384 transfer (transfers from a Nunc 384 well plate) -Falcon (destination is to a Falcon 96 well plate)
--Mix (aspirates twice in the sourse plate)
---Redoes (a procedure that allows us to transfer a quadrant at a time).
----Redo Setup (Set Up procedure)
----- Quadrant 1 pro
----- Quadrant 2 pro
----- Quadrant 3 pro
----- Quadrant 4 pro

Remember: you can hit Run Program for as many cassettes as necessary in each quadrant, but to switch quadrants, you will need to do another Set Up procedure first.

Prep Trak I has been adapted to transfer into Nunc 96 well plates as well. The procedure for this is as follows:

MT384 transfer (transfers from a Nunc 384 well sourse plate)
-Nunc (destination is to a Nunc 96 well plate)
--Mix (aspirates twice in the sourse plate)
---MT 384 N mix R1R2 SU.pro (Set Up procedure)
----MT 384 N mix R1 R2 pro. (procedure)

Procedure for loading cassettes:

96 96 96 96 96 96 96 96 96 96 96 96 96 96 96 96 96 96 96 96 96 96 96 96 96 96	96 to 96 tranfer Devel The Falcon 96 well transfer plate is loaded on the left hand side, and the corresponding Falcon 96 well destination plate is loaded parallel to it on the right hand side of the Fetcher platform.
Figure 3.

- - - - 96 96 96 96 96 96 96 96 384 384 96 96 96 96 96 96 96 96 96 96 384 384	384 to 96 transfer MT 384 F mix R1R2 pro. // MT 384 N mix R1R2 pro. For each cassette, the Nunc 384 well plate is loaded first, starting at the bottom, followed by the corresponding four Falcon 96 well destination plates. Each cassette is loaded similarly, there is no left or right preference. The same procedure is used when transfering from a Nunc 384 well plate to a Nunc 96 well plate.
Figure 4.

384 96 384 96 384 96 384 96 384 96 384 96 384 96 384 96 384 96 384 96 384 96 384 96 384 96	384 to 96 transfer Redoes All the Nunc 384 well transfer plates of the same quadrant needed are placed on the left hand side, and the corresponding Falcon 96 well destination plates are loaded parallel to them on the right hand side of the Fetcher platform.
Figure 5.