In this tutorial, I will show you how to create a simple PCR preparation protocol using the VWorks software.
This video assumes that you already know how to create a device file in the VWorks software.
If you do not have this background, please view the Device File video first before continuing.
You can also learn about device files and writing basic protocols in the VWorks Automation Control User Guide.
Before I start creating the protocol in the VWorks software, I like to outline what my protocol will do.
For my protocol, I have a 12-column reservoir, a template plate, and a PCR reaction plate.
In the reservoir the first column, or trough, contains the master mix, the second column contains the primer, and the third column contains water.
For now, the fourth column will remain empty. The remaining columns, 5 through 12, will not be used.
By the way, because I’m creating a dilution mix in the reservoir, I’ll call it the dilution plate.
In my template plate, I’ve got the DNA segments that I want to amplify.
The PCR reaction plate is empty, but will host the PCR ingredients during the protocol run.
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.
As long as I transfer them in the order I mentioned, there should be no contamination.
Then I plan to change tips and transfer some primer from column 2 to column 4.
With the tips still on, I will transfer the mixture from the dilution plate to the PCR reaction plate.
This will be done in two moves, because the total volume I want to transfer is greater than the capacity of the tips.
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.
When this part is finished, I will take the tips off.
In the second part of the protocol, I will transfer some template to the PCR reaction plate using a new set of tips.
Column 12 of the PCR reaction plate is reserved for the control, so the template will be transferred to columns 1 through 11.
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.
When I’m done, I’ll remove the tips.
So that's the overall plan.
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.
After some carefully planning, this is what I came up with.
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.
I will need two boxes of pipette tips. One box will be used for the dilution mix process.
I will place it at deck location 2. A second box will be used for the template-transfer process.
This box will be placed at deck location 9.
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.
With the workflow and deck layout planned out, I can now go into the VWorks software and start writing my protocol.
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,
and the selected profile is correct.
Everything looks good so far. So I am going to create my protocol.
I'll click File, select New, and then select Protocol.
I'll save it right now.
I’ll call it PCR Protocol, and click Save.
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.
In the Plate identity area, I'll click in the Plate name box, and type PCR Process.
Next, I will indicate the layout of the labware on the Bravo deck. To do this, I will click Configure Labware.
In the Configure Labware dialog box, I will specify the locations of the various labware on the Bravo deck that I showed you earlier.
At location 2, I will select the tipbox that I want to use for the PCR preparation process.
I will call it Tipbox for PCR Prep.
At location 5, I will select the PCR reaction plate I want to use.
I will call it PCR Reaction Plate.
At location 6, I will select the reservoir I want to use.
And I will call it Dilution Plate, because that’s where I’m creating the dilution mix.
At location 8, I will select the plate I want to use for the template.
I will call it Template Plate.
And finally, at location 9, I will select the tipbox I want to use for the template-transfer.
And I will call it Tipbox for PCR Reaction.
Now let’s make sure I’ve entered everything correctly, I'm going to check the graphic of the deck.
Everything looks good, so I will click OK to save the changes.
All of the labware I just configured appear in the protocol as expected.
If I refer back to my plan for the Bravo deck, I see that I’m missing one more thing: the tip trash.
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.
So I'm going to go to the Startup Protocol.
I will click Add Process.
and add the Configure Static Labware task.
In the Task Parameters area, I will select the Tip Trash to indicate the tip trash hole is at location 4.
In the graphic area, I see that the tip trash hole is at location 4. So everything is set for the protocol.
So I'll go back to the Main Protocol and start adding the liquid-handling tasks.
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.
So, I'll go ahead and add the first Bravo subprocess.
In the SubProcess Properties area, I’ll name it PCR Prep.
In the graphic area, I'll double check to make sure the correct device is in use.
I see that the Bravo deck has the labware I set up earlier, thus confirming that I have the correct device for the subprocess.
Now I’m ready to add tasks to this subprocess.
The first thing I must do before I can perform any liquid-handling tasks is press on pipette tips.
So I will add the Set Head Mode task to specify the pipette channels to use.
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.
I'll go ahead and click the Head mode box, where it currently says All barrels, and then click the browse button.
In the Head Mode Selector dialog box, notice that all of the channels are selected by default.
To change the channel selection, in the Subset mode list, I will select Full column.
Notice that now the right-most column is selected. This is what I want…
So I'll click OK to save the setting and return to the protocol.
Now I’m ready to add the Tips On task.
In the Tips On Properties area, I will select Tipbox for PCR Prep.
Because I want to track the tips during the protocol, I select Allow automatic tracking of tip usage.
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.
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.
In the Aspirate Properties area, I will select the Dilution Plate.
I want to aspirate 102 microliters from the column.
Next, I will select a liquid class, which I created when I set up the software.
Briefly, a liquid class defines the pipetting speed, accuracy, and precision.
For more information about liquid classes and how to create one, see the VWorks Automation Control Setup Guide.
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.
Remember that the water is in column 3, so I will select column 3, and then clear the first column.
I'll click OK to save the change.
I want to dispense the water, so I'll add the Dispense task.
I want to dispense the water in the Dilution column of the reservoir, so I'll select Dilution Plate.
I want to dispense all of the water that I picked up, which is 102 microliters.
Once again, I will select a liquid class that was already created in the Liquid Library.
I’m dispensing into the Dilution column, or column 4, so…
I'll select column 4, clear column 1,…
and click OK to save the changes.
Next, I want to transfer some master mix to the Dilution column. So I will add the Aspirate task.
I want to aspirate from the Dilution Plate, so I'll select that.
I want to aspirate 150 microliters of the master mix.
I will select the desired liquid class.
The master mix is in column 1 of the Dilution Plate.
Next, I will dispense the master mix into the Dilution column of the Dilution Plate.
I want to dispense all of the 150 microliters.
I'll select the desired liquid class.
and dispense the master mix into the Dilution column, which is column 4.
Finally, I will transfer some primer to the Dilution column.
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.
So I'll add the Tips Off task, …
discard the tips in the Tip Trash, …
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.
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.
I'm going to open the Well Selection dialog box, and change the selection to column 12.
Everything looks right for tip removal. Now I’m ready to press on new pipette tips.
I’ll go ahead and add the Tips On task.
I’m still preparing the dilution mix, so I will pick up tips from the Tipbox for PCR Prep.
I want to track tips, so I will select this option.
And I only want one column of tips, which is already selected here.
Now I’m ready to transfer the primer. First, I’ll add the Aspirate task.
I’m aspirating from the Dilution Plate, so I will select that.
I want to aspirate 24 microliters.
I'll select the liquid class.
Remember that the primer is in column 2 in the Dilution Plate, so I will select column 2.
I want to dispense the primer…
in a different column of the Dilution Plate.
I will dispense all 24 microliters I just picked up.
I will select the liquid class for the primer.
And finally, I want to dispense the primer into the Dilution column, which is column 4.
Now that I’ve got everything in the Dilution column, I can go ahead and mix.
I'll add the Mix task.
The mixing will happen in the Dilution Plate.
I will use a mix volume of 200 microliters.
I'll select the desired liquid class.
I'll mix the liquid in the Dilution column, or column 4.
OK, I’ve created the dilution mix. Now I want to transfer the dilution mix to the PCR Reaction Plate.
I'll add the Aspirate task.
I want to aspirate the mixture from the Dilution Plate.
I will select the desired liquid class.
Remember that the mixture is in the Dilution column, which is column 4 in the Dilution Plate. So I will select that column.
Oh, let me specify the volume I want to aspirate before moving on.
Now let’s dispense the mixture into the PCR Reaction Plate.
I will dispense the desired volume.
I will use this liquid class…
And I will dispense into columns 1 through 6 of the PCR Reaction Plate.
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.
So I will repeat the transfer. Add Aspirate…
I will aspirate from the Dilution plate.
… and pick the desired volume.
I'll select the desired liquid class.
Again, I will aspirate from column 4 of the Dilution Plate, …
and dispense into the PCR Reaction Plate.
I will dispense the desired volume.
I will select the desired liquid class, …
and this time, dispense into columns 7 through 12.
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.
The tips will go into the Tip Trash.
The tips are on column 12 of the pipette head, so I will select that column.
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.
I'll name the subprocess PCR Reaction so that it can be distinguished from the PCR Prep subprocess.
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.
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.
Remember that column 12 of the PCR plate will contain the control, which is the dilution mix without the template.
So when I select the channels, I want to make sure that only 11 consecutive columns are selected.
To do this, I will select Partial row/column. Notice that in the graphic, a single channel, at H12, is selected in the corner.
To select columns 2 through 12, I will click the B1 channel. The graphic now shows that columns 2 through 12 are selected.
To confirm that I have the correct columns, I'll look at the Current selection area here. And it looks right.
So I will click OK to save the changes.
I'll go ahead and add the Tips On task.
I want the tips in the Tipbox for PCR Reaction, so I will select that.
Of course, I want to track the tips, so I will select that option.
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.
Now I'll add the Aspirate task.
I'll aspirate from the Template Plate.
I will aspirate 2 microliters.
I will use the desired liquid class.
And I'll check the well selection to make sure that only columns 1 through 11 are selected. And it looks correct here.
Now, I'll dispense the template.
I'll dispense it into the PCR Reaction Plate.
I will dispense 2 microliters.
I will use the desired liquid class, …
and dispense into columns 1 through 11 only, because column 12 contains the control.
Now that we have everything we need from the Template Plate, we can go ahead and mix.
So I'll add the Mix task.
The mixing will take place in the PCR Reaction Plate.
I will specify a mix volume of 20 microliters, …
and select the desired liquid class.
I will mix in columns 1 through 11, where I added the template.
Before I add the water to column 12, I need to change tips. So I'll add the Tips Off task.
I will discard the used tips in the Tip Trash.
I don’t need to mark the tips as used.
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.
Now let’s press on new tips. Again, I’ll start with the Set Head Mode task.
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.
To change the channel selection, I will select Full column. Notice that column 12 is selected.
And I'll click OK to save the changes.
I'll go ahead and add the Tips On task.
I will use the tips from the Tipbox for PCR Reaction.
I will track the tips.
With the tips on, I can now transfer the water. I’ll add the Aspirate task.
I'll aspirate from the Dilution Plate, …
2 microliters of water.
I will use the desired liquid class.
And remember that the water is in column 3 of the Dilution Plate. So I'll select that column.
Finally, I will dispense the water …
into the PCR Reaction Plate.
I'll dispense 2 microliters of the water.
I will select the desired liquid class.
The control is in column 12, so I'll make sure that column is selected.
After the water is transferred, I can remove the tips. So I’ll add the Tips Off task.
Once again, I want to discard the used tips, so I will select the Tip Trash.
And the tips are on column 12 of the pipette head, so I'll make sure column 12 is selected for the task.
With that, I’m finished with the protocol.
I’m now ready to compile the protocol. Before I do that, I'll hide the Available Tasks area.
I'll also hide the Task Parameters area, …
and expand the PCR Prep subprocess so that we can see it.
I'm going to zoom out a bit so that we can see the entire protocol.
I'll start the compile process.
Let’s check the Main Log area. I see that I have no errors or warnings. So far so good.
So I’ll go ahead and save the protocol.
Now I'll start the simulation.
I’ll run the protocol one time, …
and start it immediately.
Here, I have to specify full tipboxes.
I'll select all wells for the PCR prep tipbox, …
and do the same for the PCR reaction tipbox.
I’m not using any barcodes, so I'll skip this step.
I don’t have any special notes about the protocol, so I’ll leave it blank.
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.
It looks like the simulation ran successfully.
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.