In the instruction video, I explained the purpose of my protocol…
…and showed how the devices and labware are set up. In this video, I will create the protocol and add the hit-picking tasks.
With my devices and labware planned out, I can start to work in the VWorks software.
First, I'm going to open the device file to make sure all of the devices I need for the protocol are in
the existing device file. Everything looks good.
Then I'm going to create my new protocol. I'll go to the File menu, select New, and then select Protocol.
I want to make sure the correct device file is associated with this protocol. So I'll click Protocol Options and check the device file path.
It looks fine, so I’ll go ahead and save the protocol file.
On the File menu, I'll click Save. I’ll call it MyHitPickProtocol, and click Save.
The first thing I should do in my new protocol is configure the Bravo deck with the hit-picking tipboxes. I will use the Configure Labware feature in this case, because each tipbox
starts out on the Bravo deck and will remain on the deck throughout the run.
Also, each is a single instance that will be used over and over again during the hit-picking process.
To learn more about configured labware and how it differs from process labware, see the VWorks Automation Control User Guide.
I'm going to click Configure Labware. In the Configure Labware dialog box, I'll select Bravo, because that’s the device on which I will add the configured labware.
Remember that I want my Tips On box, the filled tipbox, at location 2. So at location 2, I'll select the tipbox I want to use, and then type a name for it.
I’ll call it Tips On for hit-picking, or HP for short, so that I can easily identify it.
And the empty tipbox for used tips will be at location 3. So at location 3, I'll select the tipbox, and then give it a name. I’ll call it Tips Off for hit-picking (or HP).
After setting up the deck with the labware, I'll check the graphic to confirm that the labware are at the correct locations.
The graphic confirms that everything is correct. So I'll click OK to return to the protocol.
Back in the protocol, I should see the two tipboxes I just configured. And here they are, one for Tips On and one for Tips Off.
Now I’m ready to add processes. A process is a sequence of tasks that will be performed on a group of labware.
In my case, I need four processes, one for the source plates, one for the destination plate, one for the tipbox that will be used for serial dilution and plate replication,
and one for the replication plates. Which one should I add first?
I typically like to put the labware that will be involved in all of the processes throughout the protocol at the top.
In this case, it’s the destination plate process.
I'm going to make use of the existing process at the top here. I'll call it Destination so it’s easy to recognize. Then I'll select the type of plate it is.
Next, I'll click Add Process to add the source plate. I’ll call it Source so that it’s easy to recognize, and select its plate type.
I'm going to click Add Process again and add the Tipbox for serial dilution and replication.
I’ll call it Tipbox for serial dilution and replications (using abbreviations), and select the labware type.
And finally, I’ll add the plates that are the replications of the destination plate. I’ll call it Replication Plate and select the type of labware it is.
After all of the processes are in place, I can add tasks. I want to downstack the destination and source plates from the BenchCel stackers and place them on the Bravo deck.
So I'll add a Downstack task in the Destination plate process.
I'll select the device to downstack from. In this case, it’s BenchCel stacker 1.
After the plate is downstacked, it is automatically placed at Bravo deck location 9, because that’s the hand-off location specified in the device file.
But I want the plate at location 6, so I’ll add a Place Plate, select Bravo for the device, and select location 6.
Now the Bravo robot will move the plate from location 9 to 6.
I’ll add another Downstack task but in the Source plate process.
I'll select BenchCel stacker 3, because that’s where they’re stored.
And I'll add Place Plate to move the source plate from deck location 9 to location 5.
With the destination and source plates on deck, the hit-picking can start.
I’ll add a Bravo SubProcess task, name it Hit Picking so that I can easily identify it later.
Remember that in addition to the destination plate, the source plate is also involved in the hit-picking process.
So I need to add the same subprocess in the Source plate process.
The easiest way to do this is to copy the Hit Picking subprocess that is in the Destination plate process and paste it in the Source plate process.
Now I'll add the Hit Pick Replication task in the subprocess. Notice that as soon as I add it, the Hit Pick Routine Wizard opens.
The wizard will walk me through the entire setup.
On the first page of the wizard, I'll select the source plate and destination plate. I'll also select their locations on the Bravo deck.
Below the labware section, I can specify the dispense pattern in the destination plate. The wizard allows me to select an existing format file or create one.
I don’t have a format file, so I'll click Launch format wizard.
In the Hit Pick Replication Format Editor, I'll click Create new format file, type a name for the new file (I'll call it 16SampleColumnLayout), and click OK.
Back in the editor, I see the new format file name on the left.
Now I’ll click Launch format wizard to add contents to the format file.
Under Operation Type, I'll select Hit Picking. Under Labware, I'll select the Source plate type, and destination plate type.
On this next page, I must specify the dispense pattern. Remember that I want the samples to be dispensed into a single column in the destination plate.
So I will select Column-wise.
I’m going to leave the Dispense Properties for now, because I am not replicating the source samples.
In the destination plate graphic area, I have to specify which wells are available for the dispensing task.
Again, I want the samples to be dispensed in the first column of the destination plate, so I need to select column 1.
To do this, I'll right-click the first well at the top of the column, and select Clear all selected wells.
Then I'll right-click the well again and choose Select all wells in highlighted column. Now column 1 is selected.
I'll go to the next page…
I’m not performing serial dilution, so I'm going to leave the No selection for Dilution Series. I plan to transfer 30 microliter, so in the Transfer volume box, I'll type 30.
When I click Next on this page, I get a message telling me that the software might take some time to generate the format preview graphic. I’ll just click OK to continue.
On the Format Preview page, there is a graphic of the plate with different colored wells. Each color represents a unique sample.
I’m dispensing 16 different samples into the first column, so I should see 16 different colors here.
Everything looks good, so I'll click Finish. Back in the Editor, I'll click Save Changes, then close the dialog box.
Back in the Hit Pick Routine Wizard, I'll select the format I just created.
Next, I can specify the source plates I want to use and the wells from which I will transfer samples. This information is contained in an input file that I already have.
The software gives me two options: specify an input file at run time or specify the file now. I have the file already so I can specify it now.
I'll click the browse button to locate and select the file.
I see that the correct file is selected. So I’m going to move on.
On this page, I can set Aspirate and Dispense parameters. In this example, I will use the default values.
For a description of these parameters, see the VWorks Automation Control User Guide.
Next, I can specify any additional operations to be performed, such as mixing. I do want to mix, so I'll select Yes.
I want to change tips instead of washing them, so I'll select that.
And finally, remember that I configured the tipboxes on the Bravo deck when I first created the protocol.
The tipboxes are single instances in this protocol, so I'll select Never. And I'll continue to the next page…
Now I will select the pipette channels to use during the hit-picking process.
I want to use a single pipette, and it should be at position A24. So in the Subset Mode list, I'll select Single barrel.
And in the Subset orientation list, I'll select Back right.
Next, I can set the mixing parameters. I’ll go ahead and use the default parameter settings.
And finally, I will set the tip-changing parameters. I'll select the tipbox I configured earlier for the Tips On operation and select its location, which is 2.
Then I'll select the option for tip-tracking.
I'll also select the tipbox for the Tips Off operation and select its location, which is 3.
I'll select tip-tracking. The option for marking tips as used is already selected, so I’ll leave that as is. And with that, I’m finished with the wizard!
When I click Finish, the software displays a message to let me know that changes have been made to the hit-pick routine. Clicking OK will overwrite existing setup.
This is a new protocol and I’m creating a new hit-picking routine, so I'll click OK to continue.
Based on the information I provided in the wizard, the software adds the Aspirate, Dispense, Mix, tip-changing, tip-tracking, and other tasks that are necessary to produce
the desired hit-picking results. Notice that the hit-picking tasks appear in both the Destination plate process and the Source plate process.
At this point, I’m not quite done with the hit-picking part of the protocol. There is something else I have to do.
In my device setup, I do not have a barcode reader on any Bravo platepad. So as the source plates enter the Bravo device, barcodes are not scanned for verification.
I need to turn this off in the software, or I'll get errors.
To turn off barcode verification …
I'll select the Hit Pick task in the routine. In the Task Parameters area, I'll look for Verify source plate barcodes, and clear that check box.
Now I’m finished with the hit-pick routine.