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In this tutorial, I will show you how to create a simple PCR preparation protocol using the VWorks software.

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This video assumes that you already know how to create a device file in the VWorks software.

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If you do not have this background, please view the Device File video first before continuing.

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You can also learn about device files and writing basic protocols in the VWorks Automation Control User Guide.

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Before I start creating the protocol in the VWorks software, I like to outline what my protocol will do.

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For my protocol, I have a 12-column reservoir, a template plate, and a PCR reaction plate.

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In the reservoir the first column, or trough, contains the master mix, the second column contains the primer, and the third column contains water.

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For now, the fourth column will remain empty. The remaining columns, 5 through 12, will not be used.

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By the way, because I’m creating a dilution mix in the reservoir, I’ll call it the dilution plate.

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In my template plate, I’ve got the DNA segments that I want to amplify.

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The PCR reaction plate is empty, but will host the PCR ingredients during the protocol run.

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In the first part of the protocol, I will transfer some water and then some master mix to column 4 of the dilution plate using the same pipette tips.

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As long as I transfer them in the order I mentioned, there should be no contamination.

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Then I plan to change tips and transfer some primer from column 2 to column 4.

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With the tips still on, I will transfer the mixture from the dilution plate to the PCR reaction plate.

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This will be done in two moves, because the total volume I want to transfer is greater than the capacity of the tips.

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The first transfer will dispense the mixture into columns 1 through 6 of the PCR reaction plate. The second transfer will dispense the mixture into columns 7 through 12.

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When this part is finished, I will take the tips off.

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In the second part of the protocol, I will transfer some template to the PCR reaction plate using a new set of tips.

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Column 12 of the PCR reaction plate is reserved for the control, so the template will be transferred to columns 1 through 11.

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Instead of the template, I want to add water to column 12. Using a new set of tips, I will transfer some water from the dilution plate to column 12 of the PCR reaction plate.

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When I’m done, I’ll remove the tips.

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So that's the overall plan.

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I plan to use my Bravo Platform to run the protocol. So I need to figure out how to place the various labware on the deck.

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After some carefully planning, this is what I came up with.

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I will place the PCR reaction plate at the center of the deck, at location 5, the reservoir, or dilution plate at location 6, and the template at location 8.

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I will need two boxes of pipette tips. One box will be used for the dilution mix process.

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I will place it at deck location 2. A second box will be used for the template-transfer process.

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This box will be placed at deck location 9.

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Finally, I need a place to discard the used tips. My Bravo Platform came with a tip trash hole at deck location 4. So that’s where the used tips will go.

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With the workflow and deck layout planned out, I can now go into the VWorks software and start writing my protocol.

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In the VWorks software, the first thing I want to do is check that the device file I created earlier is OK. I see that I’ve got the Bravo device here, 

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and the selected profile is correct.

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Everything looks good so far. So I am going to create my protocol.

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I'll click File, select New, and then select Protocol.

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I'll save it right now.

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I’ll call it PCR Protocol, and click Save.

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This is a simple protocol with only one process, so I’ll go ahead and name the process now. I'll call it PCR Process.

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In the Plate identity area, I'll click in the Plate name box, and type PCR Process.

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Next, I will indicate the layout of the labware on the Bravo deck. To do this, I will click Configure Labware.

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In the Configure Labware dialog box, I will specify the locations of the various labware on the Bravo deck that I showed you earlier.

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At location 2, I will select the tipbox that I want to use for the PCR preparation process.

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I will call it Tipbox for PCR Prep.

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At location 5, I will select the PCR reaction plate I want to use.

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I will call it PCR Reaction Plate.

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At location 6, I will select the reservoir I want to use.

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And I will call it Dilution Plate, because that’s where I’m creating the dilution mix.

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At location 8, I will select the plate I want to use for the template.

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I will call it Template Plate.

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And finally, at location 9, I will select the tipbox I want to use for the template-transfer.

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And I will call it Tipbox for PCR Reaction.

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Now let’s make sure I’ve entered everything correctly, I'm going to check the graphic of the deck.

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Everything looks good, so I will click OK to save the changes.

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All of the labware I just configured appear in the protocol as expected.

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If I refer back to my plan for the Bravo deck, I see that I’m missing one more thing: the tip trash.

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The tip trash hole is a permanent structure on the Bravo deck. And permanent locations are specified using the Configure Static Labware task in the Startup Protocol.

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So I'm going to go to the Startup Protocol.

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I will click Add Process.

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and add the Configure Static Labware task.

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In the Task Parameters area, I will select the Tip Trash to indicate the tip trash hole is at location 4.

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In the graphic area, I see that the tip trash hole is at location 4. So everything is set for the protocol.

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So I'll go back to the Main Protocol and start adding the liquid-handling tasks.

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In the protocol, I will use two Bravo subprocesses. The first subprocess will create the dilution mix. The second subprocess will add the template to the PCR reaction plate.

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So, I'll go ahead and add the first Bravo subprocess.

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In the SubProcess Properties area, I’ll name it PCR Prep.

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In the graphic area, I'll double check to make sure the correct device is in use.

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I see that the Bravo deck has the labware I set up earlier, thus confirming that I have the correct device for the subprocess.

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Now I’m ready to add tasks to this subprocess.

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The first thing I must do before I can perform any liquid-handling tasks is press on pipette tips.

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So I will add the Set Head Mode task to specify the pipette channels to use.

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Remember that I want to transfer liquid from and to single columns in the reservoir, so I need to specify a single column of channels.

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I'll go ahead and click the Head mode box, where it currently says All barrels, and then click the browse button.

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In the Head Mode Selector dialog box, notice that all of the channels are selected by default.

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To change the channel selection, in the Subset mode list, I will select Full column.

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Notice that now the right-most column is selected. This is what I want…

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So I'll click OK to save the setting and return to the protocol.

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Now I’m ready to add the Tips On task.

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In the Tips On Properties area, I will select Tipbox for PCR Prep.

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Because I want to track the tips during the protocol, I select Allow automatic tracking of tip usage.

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Notice here that a single column was selected for the tips. This is because of the pipette channel selection I made in the Set Head Mode task.

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Now I’m ready to aspirate water from the third column of the dilution plate and dispense it into the dilution column. So I'll add the Aspirate task.

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In the Aspirate Properties area, I will select the Dilution Plate.

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I want to aspirate 102 microliters from the column.

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Next, I will select a liquid class, which I created when I set up the software.

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Briefly, a liquid class defines the pipetting speed, accuracy, and precision.

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For more information about liquid classes and how to create one, see the VWorks Automation Control Setup Guide.

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And finally, before I leave this Aspirate task, I have to select the column from which to aspirate. So I'll click Well selection, and then click the browse button.

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Remember that the water is in column 3, so I will select column 3, and then clear the first column.

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I'll click OK to save the change.

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I want to dispense the water, so I'll add the Dispense task.

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I want to dispense the water in the Dilution column of the reservoir, so I'll select Dilution Plate.

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I want to dispense all of the water that I picked up, which is 102 microliters.

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Once again, I will select a liquid class that was already created in the Liquid Library.

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I’m dispensing into the Dilution column, or column 4, so…

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I'll select column 4, clear column 1,…

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and click OK to save the changes.

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Next, I want to transfer some master mix to the Dilution column. So I will add the Aspirate task.

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I want to aspirate from the Dilution Plate, so I'll select that.

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I want to aspirate 150 microliters of the master mix.

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I will select the desired liquid class.

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The master mix is in column 1 of the Dilution Plate.

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Next, I will dispense the master mix into the Dilution column of the Dilution Plate.

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I want to dispense all of the 150 microliters.

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I'll select the desired liquid class.

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and dispense the master mix into the Dilution column, which is column 4.

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Finally, I will transfer some primer to the Dilution column.

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Before I do that, I have to change pipette tips. The tips I’ve used so far have touched water and the master mix. I cannot use the same tips to transfer the primer.

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So I'll add the Tips Off task, …

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discard the tips in the Tip Trash, …

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and clear the check box for Mark tips as used. Actually, I don’t really need to clear the check box, because by default, you cannot track tips after they are discarded.

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Remember that the pipette tips are in column 12 of the pipette head. But the Well selection for the Tips Off task shows that column 1 is selected. So I need to fix it.

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I'm going to open the Well Selection dialog box, and change the selection to column 12.

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Everything looks right for tip removal.  Now I’m ready to press on new pipette tips.

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I’ll go ahead and add the Tips On task.

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I’m still preparing the dilution mix, so I will pick up tips from the Tipbox for PCR Prep.

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I want to track tips, so I will select this option.

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And I only want one column of tips, which is already selected here.

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Now I’m ready to transfer the primer. First, I’ll add the Aspirate task.

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I’m aspirating from the Dilution Plate, so I will select that.

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I want to aspirate 24 microliters.

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I'll select the liquid class.

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Remember that the primer is in column 2 in the Dilution Plate, so I will select column 2.

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I want to dispense the primer…

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in a different column of the Dilution Plate.

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I will dispense all 24 microliters I just picked up.

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I will select the liquid class for the primer.

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And finally, I want to dispense the primer into the Dilution column, which is column 4.

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Now that I’ve got everything in the Dilution column, I can go ahead and mix.

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I'll add the Mix task.

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The mixing will happen in the Dilution Plate.

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I will use a mix volume of 200 microliters.

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I'll select the desired liquid class.

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I'll mix the liquid in the Dilution column, or column 4.

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OK, I’ve created the dilution mix. Now I want to transfer the dilution mix to the PCR Reaction Plate.

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I'll add the Aspirate task.

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I want to aspirate the mixture from the Dilution Plate.

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I will select the desired liquid class.

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Remember that the mixture is in the Dilution column, which is column 4 in the Dilution Plate. So I will select that column.

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Oh, let me specify the volume I want to aspirate before moving on.

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Now let’s dispense the mixture into the PCR Reaction Plate.

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I will dispense the desired volume.

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I will use this liquid class…

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And I will dispense into columns 1 through 6 of the PCR Reaction Plate.

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So far, I’ve only transferred the mix into columns 1 through 6 as shown here. I need to fill in columns 7 through 12.

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So I will repeat the transfer. Add Aspirate…

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I will aspirate from the Dilution plate.

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… and pick the desired volume.

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I'll select the desired liquid class.

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Again, I will aspirate from column 4 of the Dilution Plate, …

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and dispense into the PCR Reaction Plate.

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I will dispense the desired volume.

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I will select the desired liquid class, …

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and this time, dispense into columns 7 through 12.

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Before I start the next part of the protocol, I must discard the used pipette tips. So I’ll go ahead and add the Tips Off task.

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The tips will go into the Tip Trash.

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The tips are on column 12 of the pipette head, so I will select that column.

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I’ve just finished the dilution mix tasks. Now I’m ready to add the template to the PCR reaction plate, so I will add a new Bravo subprocess.

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I'll name the subprocess PCR Reaction so that it can be distinguished from the PCR Prep subprocess.

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Before I add new tasks, let me collapse the PCR Prep subprocess so that I can easily see the tasks for the PCR Reaction subprocess.

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The first thing I want to do in this subprocess is press on new pipette tips. So I will add the Set Head Mode task and select the channels I want to use.

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Remember that column 12 of the PCR plate will contain the control, which is the dilution mix without the template.

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So when I select the channels, I want to make sure that only 11 consecutive columns are selected.

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To do this, I will select Partial row/column. Notice that in the graphic, a single channel, at H12, is selected in the corner.

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To select columns 2 through 12, I will click the B1 channel. The graphic now shows that columns 2 through 12 are selected.

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To confirm that I have the correct columns, I'll look at the Current selection area here. And it looks right.

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So I will click OK to save the changes.

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I'll go ahead and add the Tips On task.

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I want the tips in the Tipbox for PCR Reaction, so I will select that.

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Of course, I want to track the tips, so I will select that option.

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Notice that the Well selection is grayed out. I already selected the channels in the Set Head Mode task, so I don’t need to do it again here.

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Now I'll add the Aspirate task.

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I'll aspirate from the Template Plate.

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I will aspirate 2 microliters.

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I will use the desired liquid class.

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And I'll check the well selection to make sure that only columns 1 through 11 are selected. And it looks correct here.

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Now, I'll dispense the template.

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I'll dispense it into the PCR Reaction Plate.

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I will dispense 2 microliters.

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I will use the desired liquid class, …

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and dispense into columns 1 through 11 only, because column 12 contains the control.

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Now that we have everything we need from the Template Plate, we can go ahead and mix.

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So I'll add the Mix task.

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The mixing will take place in the PCR Reaction Plate.

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I will specify a mix volume of 20 microliters, …

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and select the desired liquid class.

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I will mix in columns 1 through 11, where I added the template.

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Before I add the water to column 12, I need to change tips. So I'll add the Tips Off task.

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I will discard the used tips in the Tip Trash.

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I don’t need to mark the tips as used.

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I have to select which tips to be tossed. Remember that the tips are on columns 2 through 12 of the pipette head, so I will select those columns.

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Now let’s press on new tips. Again, I’ll start with the Set Head Mode task.

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This time, I will aspirate from a single column, column 4, of the Dilution Plate. So I need to select 1 column of channels, not all 12 columns.

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To change the channel selection, I will select Full column. Notice that column 12 is selected.

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And I'll click OK to save the changes.

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I'll go ahead and add the Tips On task.

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I will use the tips from the Tipbox for PCR Reaction.

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I will track the tips.

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With the tips on, I can now transfer the water. I’ll add the Aspirate task.

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I'll aspirate from the Dilution Plate, …

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2 microliters of water.

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I will use the desired liquid class.

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And remember that the water is in column 3 of the Dilution Plate. So I'll select that column.

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Finally, I will dispense the water …

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into the PCR Reaction Plate.

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I'll dispense 2 microliters of the water.

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I will select the desired liquid class.

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The control is in column 12, so I'll make sure that column is selected.

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After the water is transferred, I can remove the tips. So I’ll add the Tips Off task.

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Once again, I want to discard the used tips, so I will select the Tip Trash.

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And the tips are on column 12 of the pipette head, so I'll make sure column 12 is selected for the task.

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With that, I’m finished with the protocol.

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I’m now ready to compile the protocol. Before I do that, I'll hide the Available Tasks area.

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I'll also hide the Task Parameters area, …

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and expand the PCR Prep subprocess so that we can see it.

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I'm going to zoom out a bit so that we can see the entire protocol.

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I'll start the compile process.

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Let’s check the Main Log area. I see that I have no errors or warnings. So far so good.

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So I’ll go ahead and save the protocol.

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Now I'll start the simulation.

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I’ll run the protocol one time, …

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and start it immediately.

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Here, I have to specify full tipboxes.

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I'll select all wells for the PCR prep tipbox, …

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and do the same for the PCR reaction tipbox.

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I’m not using any barcodes, so I'll skip this step.

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I don’t have any special notes about the protocol, so I’ll leave it blank.

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When I click Finish, the simulation starts. I can follow the green dot in the protocol area to see that the protocol is running as planned.

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It looks like the simulation ran successfully.

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Now I can perform a dry run with empty plates to make sure it works in real life before running it using my valuable samples and reagents.

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