BioGrid Stamping Protocol

Pour 250 mL LB agar + Chloramphenicol (20mg/mL) into 20x20 cm trays and let cool.

Label using pencil, each of the six panels on each filter with the filter number and an arrow pointing to the end of the filter with the first panel. This way when the panels are cut, the orientation will still be decipherable.

Place +charged nylon membranes (Amersham RPN2250B) over the agar. You want to be sure the filter is centered on the agar so the clones are not stamped off the edges. Avoid air bubbles between the membrane and the agar by taking two corners with forceps and letting the middle touch the agar first and roll the rest on to the surface.

Move the following instruments to the zero position

Transfer arm in X -the far right side of the machine

Transfer arm in Y -the highest position

Filter trays 1-4 -as far back as possible

BioBank holder bar -as far down as possible

Vacuum lid lifter -as far down as possible

Source plate hooks -as far forward as possible

Start up the BioGrid by pressing the switch on the right side towards the back for the whole robot and turning the knob on for the fan towards the front on the right side

Press the red button on the right side towards the front of the when done and the machine will run through a check.

The Main Menu will appear click on the box to “Load previously saved parameters”

The program we have for stamping the filters in a 4X4 grid is “grid1”

Then click on “Set parameters”

You will see the data for the grid1 program. On the destination (right) side of the screen, select “double offset” and then select “User program offsets”

This will take you to the screen where you can input the 4 x 4 stamping pattern that can be filled in according to the pattern that you want to stamp. The stamping pattern for CHORI, the one we use, is below. This needs to be saved under the “File” menu and then you can exit. 2 6 2 3

1 4 7 5

7 8 3 6

1 5 4 8

This will take you back to the parameters page where you can finish changing the run to your specifications. When completed click on the red “OK” button at the bottom right of the screen.

Now you will be back at the Main menu. The top option is “Go”, when selected will run the setup for the program you just set the parameters for.

The computer prompts you to load the agar trays one at a time and press the interrupt button when finished. Remove the lid and load the tray into the holder with the arrows pointing toward the BioBank. Be sure the plate remains in place by lightly turning the little black screw in the front of the tray holder. Only a half turn past when it touches the tray.

The computer prompts you to load the Bio-bank. Place the 384-well plates in with A1 facing in the back right in the positions and press the interrupt button.

The computer prompts you to attach the 384-pin tool to the arm holder. Secure the tool on by tightening the little black screw really well and then press the interrupt button.

The computer will prompt you to fill the bath trays up to the step. The first tray on the left fill with dH₂O and the second fill with 80% ethanol and then press the interrupt button.

This time the machine will start the run automatically when you press the interrupt button starting with a wash cycle.

Should there be a problem or you need to change some parameter during the run just press the yellow button and hold it in and the run will be interrupted at the completion of the movement. The main menu will be on the sreen and if you need to change something slelect the “Change run parameters” and change whatever it is and then click “OK”. You can rejoin the run on the main menu then by selecting the “Rejoin run” option and it will pick up at the same place it left off.

Place lids back on the agar plates and stack the plates.

Wrap the stack of plates with Saran wrap to prevent evaporation and place upsidedown in a 37°C incubator overnight