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Setting up a hanging drop experiment with the NT8 involves six basicf steps:
When you set up a hanging drop experiment with the NT8, you must be careful to set up the dispense in the opposite direction by clicking the dispense direction arrow. This is because you will flip the plate when you put the plate seal on the MRC 2 Well Crystallization Plate. We've highlighted this step with a red text in the steps that follow.
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.
Place the LV tip caddy in its station and the MRC 2 Well Crystallization Plate on microplate station 2. Each well of the MRC 2 plate should contain about 50 μl of solution.
Tear off the tab at the angled side of the plate seal. Then, carefully remove the liner from the entire seal.
Dab a small amount of 70% Isopropanol on the reference plate. This helps adhere the plate seal to the reference plate during the dispense.
Using the crosshairs to align the plate seal to the reference plate, fasten the UVP Hanging Drop MRC Plate Seal to the microplate.
Important: The angled corners of the plate seal should be at the right side of the reference plate. After the dispense, you will flip the reference plate horizontally to the right to place the plate seal on the experiment plate.
Place the reference plate on a microplate station (angled corners of plate seal should be at the right).
Place the protein strip on the protein block, and put the protein strip clamp on top of the protein strip to hold it in place. You will pipette your protein into the protein block after you set up the software.
A hanging drop experiment template is pre-installed with your software. You may use it if you plan to combine 100 nL of protein with 100 nL of well solution for a 200 nL dispense. If you choose to use this template, you must also use the MRC 2 Well Crystallization Plate and the UVP Hanging Drop Plate Seal.
Click the Open Task List button 
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Open the Sample folder and select the Hanging Drop MRC2 100 nL Protein And 100 nL Well Solution task list.
Click Open.
Select the MRC 2 Well Crystallization Plate from the Plate Type Library and drag it onto the associated plate station in the software.
Select the UVP Hanging Drop Plate Seal from the Plate Type Library and drag it onto the associated plate station in the software.
Drag the LV Tip Caddy from the Plate Type Library to the LV Tip Caddy station in the software.
Right-click the Protein Station image and select Set Protein Volume. Enter the total protein volume for the column under the Volume field (the volume should be the same for every well in a column).
Double-click the LV Tip Caddy image to add the Pick Tip command to the task list.
Click the Repeat button 
on the Task List to start the beginning of tasks that will be repeated on every column of a plate.
Double-click the Wash Station image to add the Wash Tip command to the task list.
Double-click the Protein Block image to open the Task Details dialog box.
Next to Action, select Aspirate.
Next to Column, select the column where your protein will be.
Next to Volume, enter the volume to be aspirated.
Select a Liquid Class from the liquid class list.
Click Add.
Double-click the plate image to open the Task Details dialog box.
Next to Action, select Aspirate.
Next to Column, click column 1, then press and hold the Shift key and click column 12 to select all columns.
Next to Volume, enter the volume you wish to aspirate from the well.
Verify that the well is selected in the Select Target image. If selected, it is highlighted in yellow, as shown at right. If not, click the well to select it.
Select a Liquid Class from the liquid class list.
Next to Z-Offset, select the height you want the pipette tips to stop at before aspirating from the well.
Click Add.
Double-click the UVP Hanging Drop Plate Seal image to open the Task Details dialog box.
Verify that Dispense is selected next to Action.
Important: Click the arrow above the plate grid to reverse the dispense direction so that the arrow points to the left.
Verify that columns 1-12 are selected next to Column (if selected, they appear in dark blue). If not, click column 1, then press and hold the SHIFT key and click column 12 to select all columns.
Next to Volume, enter the desired dispense volume (generally the combined volume of the well solution and the protein volume).
Optional: Next to Mixing, select the number of mixing cycles you want the NT8 to perform before dispensing drops.
Optional: Next to Z-Offset, you may choose to leave Flexible Fingers enabled (indicated by the FF symbol), or you may click the FF button and then manually enter the distance the pipette tips should travel before dispensing your drops. It is strongly recommended that you leave Flexible Fingers enabled for this type of dispense.
Select a Liquid Class from the liquid class list.
Click Add.
Click End Repeat in the Task List to select it.
Double-click the Waste Station to add the Dispose Tip command to the Task List.
Optional: Save your Task List by clicking the Save button 
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Using a hand pipette, pipette the protein to be used in the hanging drop experiment into the protein block.
Click the Run Task List button 
to execute the Task List. The NT8 head moves to the LV Tip Caddy, picks up a column of tips, then washes them at the wash station. Next, the NT8 will aspirate protein from the protein block, then aspirate solution from the microplate well. Finally, the NT8 will dispense the hanging drops to the UVP Hanging Drop Plate Seal. This series of actions will be repeated on each column specified. Finally, the NT8 will dispose the used tips into the waste station.
When the dispense has completed, take the microplate and place it on the HD seal jig transfer. The A1 position should be at the top-left corner.
Place the UVP Seal and the reference plate on the microplate by carefully flipping it over horizontally above the microplate. Use the three steel pins to guide the reference plate into place. The angled edges of the UVP Seal should match the angled edges of the microplate. Once in place, press down firmly.
Carefully slide the reference plate to the right. The adhesive surface on the UVP Seal should keep it in place while you remove the reference plate.
Use a microplate seal roller to secure the UVP seal to the microplate surface to assure each well is properly sealed.
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NTC-V28R016
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