LCP Experiments

Given the nature of an LCP experiment, the set up for an LCP dispense requires more hardware setup than other experiments. This tutorial walks you through hardware setup, provides two options for setting up the dispense in the software, running the LCP dispense, and finally attaching the LCP coverslip to the LCP base plate.

Set Up the NT8 Hardware

Prepare the LCP syringe (for help with using the LCP mixer, see Mixing LCP Drops).
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.
Prepare the LCP Plate:

Spray the LCP plate with a duster to remove any contaminants.
Peel off the brown packing tape.
Apply 2-3 drops of 70% Isopropanol to the reference plate surface.
Aligning the four corners drop locations to the crosshairs on the reference plate and affix the LCP plate to the reference plate.

Attach the LCP syringe to the NT8’s head.

Using your left hand, align the syringe with the syringe bracket and hold it in place.
Using your right hand, move the joystick to align the plunger lock on the NT8 with the top of the plunger.
Using your left hand, place the top of the plunger into the plunger lock, and gently press the barrel of the syringe into the syringe bracket until it snaps into place.
Once in place, tighten the plunger lock thumbscrew (located directly above the plunger lock).

Tip: For additional help, see Installing LCP Attachments.

Use the lever to dispense a small amount of LCP sample from the syringe to prevent clogging and remove any air from the needle. Wipe the tip of the LCP syringe with a chem wipe.
The sensors on the NT8 will detect the presence of the LCP syringe. A dialog box will open asking if you want to calibrate the syringe. Click Yes.
Select the size of your LCP syringe and click OK. The NT8 will start calibrating the LCP syringe.

Option 1: Set Up the Software Using the LCP Template

An LCP dispense template is preinstalled with your software. You may use it if you plan to combine 800 nL well solution with 50 nL LCP drops. If you choose to use this template, you must also use the MRC 2 Well Crystallization plate as your well solution source.

To use this template:

Click the Open Task List button .
Open the Sample folder and select the LCP 50 nL Monolein And 800 nL Well Solution task list.
Click Open.
Run your experiment by clicking the Run Task List button .

Option 2: Manually Set Up the Software

Drag the LCP Plate Adapter from the Plate Type library to Microplate Station 1.
Find the plate type you used for your well solution and drag the plate to Microplate Station 2.
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 that will be repeated for every column.
Add the Wash Tip command to the Task List by double-clicking the Wash Station image.
Instruct the NT8 to aspirate well solution:

Double-click the image of the plate containing your well solution 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 want to aspirate from each well.
Click the well location in the Select Target image to select it (example at right).
Select a Liquid Class from the liquid class list.
Next to Z-Offset, enter the distance from the bottom of the well you want the pipette tip to aspirate from. The default is 0.5 mm.
Click Add.

Instruct the NT8 to dispense the LCP sample:

Double-click the LCP Plate Adapter image to open the Task Details menu.
In the Select Head image, click the LCP head to select it. The selected head is highlighted in yellow.
Next to Action, select Dispense.
Next to Column, click column 1, then press and hold the SHIFT key and click column 12 to select all columns.
Next to Stock Name, select the name of your LCP sample.
Next to Volume, select the amount of LCP sample you want to dispense to each drop from the list.
Select a Liquid Class from the liquid class list.
Click OK.

Instruct the NT8 to dispense the well solution to the drop:

Double-click the image of the LCP Plate to open the Task Details menu.
Next to Action, select Dispense.
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 of solution you want to dispense. This should match the volume you entered in step 7.
Next to Mixing, enter the number of times you want the NT8 to mix the LCP sample and well solution before dispensing.
Next to Z-Offset, enter the distance from the plate you want the pipette tips to dispense from.

OR

Click the FF button to dispense directly to the plate surface.

Click End Repeat in the Task List to select it.
Double-click the Waste Station image to add the Dispose Tip command to the Task List.

Run the LCP Dispense

Click the Run Task List button to execute the Task List. The NT8 picks up the LV tips, washes them, aspirates from the microplate containing the well solution, then dispenses the LCP sample across a column and then dispenses a column of well solution to the LCP sample drops. This process repeats for each column you specified for the LCP plate. After the NT8 has completed the tasks for each column, it disposes the tips in the waste station.

Attach the LCP Coverslip

Gently place the coverslip on the dispensed LCP plate. Use a roller or spatula to apply force to the coverslip to affix the coverslip to the base plate.