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Sitting Drop Experiments

Follow the steps below to set up and run a Sitting Drop experiment. There are five overall steps:

Turning On the NT8
Setting up the hardware
Setting up the software
Adding protein to the protein block
Running the task list you created

Power On the NT8 and Start the Software

Connect the NT8 to your computer using the USB cable.
Find the power switch located on the back-right of the NT8 and turn it on.
Double-click the NT8 icon on your computer’s desktop. It will take a few moments for the NT8 software to load while the NT8 initializes. The instrument indicator on the bottom-left corner of the software will turn green when the NT8 is connected and ready to use.

Set Up the NT8 Hardware

Place the tip caddy and the MRC 2 Well Crystallization plate on the NT8. Each well should contain 50 μL of solution.
Place the protein tray on the protein block, and put the protein tray clamp on top of the protein tray to hold it in place. You will pipette your protein into the protein block after you set up the software.

Option 1: Set up the Software with the Sitting Drop Template

A Sitting Drop experiment setup template is pre-installed with your software. You may use it if you plan to combine 100 nL of well solution with 100 nL of protein for a total dispense volume of 200 nL. You can also modify these volumes by double-clicking the item on the task list. However, you must use the MRC 2 Well Crystallization plate in order to use this template.

To use this template:

Click the Open Task List button .
Open the Sample folder and select the Sitting Drop MRC2 100 nL Protein and 100 nL Well Solution task list.
Click Open.
Set the protein volume by right-clicking the protein block image and selecting Set Protein Volume. Select the column that you will pipette protein into and enter the volume in the appropriate field. The volume must be the same in each well in a column.
Optional: If you need to change the aspirate or dispense volumes, double-click the task in the Task List and edit the volume.
Run your experiment by clicking the Run Task List button .

Option 2: Manually Set Up the NT8 Software

Mimic the NT8’s physical setup with the software:
- Select each microplate you have stationed on the NT8 from the Plate Type Library, and drag and drop them into the appropriate location in the software.
- Drag and drop the LV Tip Caddy from the Plate Type Library to the LV Tip Caddy station.
- Right-click the protein block image and select Set Protein Volume. Type in the volume of protein available in each well of each column in the tray that you plan to use.
Add the Pick Tip command to the Task List by double-clicking the LV Tip Caddy image.
Click the Repeat button on the Task List to mark the beginning of tasks you will repeat on each column of your plate.
Add the Wash Tip command to the Task List by double-clicking the Wash Station image.
Now you’ll begin adding aspirate and dispense commands. First, tell the NT8 to aspirate from your protein source by adding an Aspirate command.
- Double-click the Protein Block image.
- Next to Action, select Aspirate. Next to Column, select the column you want to aspirate the protein from. Next to Volume, type in the volume you want to aspirate.
- Click OK.
Now you’ll tell the NT8 to aspirate your well solution.
- Double-click the microplate image to open the Task Details dialog box.
- Next to Action, click Aspirate.
- Next to Column, select the columns you want to aspirate from, and enter the volume to be aspirated. To select multiple columns, hold down the SHIFT key and click the columns you want to select.
- Click the well area in the Select Target picture to select it (highlighted in yellow at right).
- Select a Liquid Class from the liquid class list.
- If you are finished adding commands for that plate, click Add. If you want to dispense to drop locations on this plate, click Add More.
Instruct the NT8 to dispense the protein and well solution mixture to the drop location on the microplate.
- If you clicked Add More in the last step, the Task Details dialog box remained open. If not, double-click the microplate image again to open it.
- Next to Action, click Dispense.
- Next to Column, select the columns you want to dispense to. Next to Volume, enter the volume to be dispensed. If you want the NT8 to mix your solution, enter the desired number of mixing cycles next to Mixing. Mixing is not required for dispense.
- Select a Liquid Class from the liquid class list.
- Next to Z-Offset, you may choose to let the NT8’s Flexible Fingers determine the dispense height (recommended), or manually specify the distance you want the fingers to stop at before dispensing.
- Click the drop location in the Select Target area. In the image on the right, drop 1 is selected.
Click End Repeat in the Task List to select it.
Add the Dispose Tip command to the Task List by clicking the Waste Station image.
Optional: Save your Task List by clicking the Save button .

Add Protein to the Protein Block

Using a hand pipette, pipette the protein to be used in the sitting drop experiment into the protein block.

Run the Task List

Click the Run Task List button to execute the Task List. The NT8 picks up the LV tips, washes them, aspirates from the protein block, aspirates from the wells in the microplate, and then dispenses to the drop location. This process repeats for each column you specified earlier. After the NT8 has completed the tasks for each column, it disposes the tips in the waste station.