In the Hit Picking video, I showed you how to include hit-picking tasks in a protocol. Now I’ll show you how to add serial dilution tasks in the protocol.
Before I do that, let’s briefly review what we’ve done so far in the protocol.
In the Hit Picking video, I added tasks to pick two or three samples from a set of six source plates and dispensed them into the first column of the destination plate.
Now I need to dilute the column serially in the destination plate.
Let’s also check where things are at the devices. At the end of the hit-pick routine, all source plates are put away at BenchCel stacker 4.
The destination plate remains at Bravo deck location 6.
What I want to do now is fill the destination plate with diluent. To do this, I will move the destination plate to the Multidrop device, and then move it back to the Bravo
device. Again, I’m moving the plate to location 5 instead of 6 because it is an accessible location for the left-most channels on the Bravo pipette head.
After adding the diluent, I can add the serial dilution tasks.
While the destination plate remains at Bravo deck location 5, I will downstack some tips from BenchCel stacker 5 and place it at Bravo deck location 7.
OK, let’s return to the protocol and add the diluent in the destination plate. I want to fill columns 2 through 24 of the destination plate with a diluent in preparation for
serial dilution. This will be done at the Multidrop device, so I need to add the Multidrop task.
Let’s take a look at the task parameters. I’ll leave Prime volume at 50 microliters. I want to dispense 30 microliters of diluent.
And I’ll leave these parameters at their default values and selections. For Plate pattern, I’m going to choose Selected columns, because I only want to dispense into columns 2
through 24. Then I'll click the next row, and click the browse button that appears to select the desired columns.
In the Well Selection dialog box, I see that only the first column is selected. To change this, I'll right-click the A2 well, and then choose Select all wells.
Then I'll click the A1 well to clear the A1 column.
I want to verify that the correct columns are selected, so I scroll to the right to see all of the columns. And it looks correct.
I’ll click OK to go back to the protocol and check that the well-column selection is reflected in the Task Parameters area.
I’m going to leave the rest of the parameters. And I’m done with this task.
Now I want to start adding tasks for the serial dilution part of this protocol. But before I can do that, I need to get things ready.
First I’ll add a Spawn Process task. The reason I’m doing this is because I want to hold those tipboxes and their associated tasks until I’m ready for them.
In the Spawn Process Task Parameters area, I'll select Tipbox for Serial Dilution and Replication.
I don’t need to select the Spawn as subroutine option, because the Tipbox process will not be a subroutine of the Destination process.
The other thing I have to do is move the Destination plate from location 6 to location 5. The reason I’m doing that is because serial dilution will be performed column-wise, and
the pipette channels I’m using are in column A1. The pipette head cannot access Destination well columns 2 through 24 if the plate remains at location 6.
For detailed information about accessible deck locations for different pipette channel selections, see the VWorks Automation Control User Guide.
But wait! Isn’t the Source plate occupying location 5? Remember from my plan that as soon as a plate is finished with its process, it will be upstacked.
Although I haven’t added that task just yet, I know in my mind that the Source plate will be moved, and location 5 will be available.
So the Destination plate can use that location for the serial dilution tasks.
When the Tipbox process is spawned, the first thing that should happen is the tipbox will be downstacked from the BenchCel stacker.
So I'll add the Downstack task in the Tipbox process.
The tipbox will be coming from BenchCel stacker 5.
And according to the plan, it should be placed at deck location 7 for the serial dilution and replication tasks.
Now I’m ready to add the serial dilution tasks. First, I'll add a Bravo Subprocess in the Destination plate process and name it Serial Dilution.
Because the tipbox is also used in the serial dilution tasks, I need to include it in the subprocess.
I'll copy the Serial Dilution subprocess and paste it in the Tipbox process. Now anything added in the subprocess in one will automatically be added in the other.
Let’s start to add the tasks in this subprocess.
Like any liquid-handling subprocess, the first thing I have to do is specify the pipette channels I want to use. I'll add the Set Head Mode task…
I'll select the channels… I'll select Full column, and then Back left, because I want the A1 column of channels. I'll go back to the protocol.
Let’s press on the tips. I'll add the Tips On task.
In the Task Parameters area, I'll select the Tipbox.
I can leave the well selection at A1, because the software will refer to the channel selection in the Set Head Mode task.
Now I’ll add the Serial Dilution task.
The serial dilution will be performed in the Destination plate, so I'll select it.
I'll click Launch serial dilution wizard, which will walk me through the setup process.
In Step 1, I get to specify how I want the liquid transfer to occur and how much.
I want the dilution to be based on transfer volume, which is already selected, so I'll move on.
Here, I can specify the transfer volume. I want to transfer a fixed volume, which is already selected. And I want to transfer 30 microliters, so I'll type that in the box.
In Step 2, I can specify additional operations. For example, I want to mix the well contents, so I'll leave that selection as is.
I don’t want to specify other operations, so I’ll leave the No additional operations selection and move on.
For an explanation of these other options, see the VWorks Automation Control User Guide.
In Step 3, I can specify the column that contains the initial concentration. Column A1 is already selected, which is correct, so I'll continue without making any changes.
Next, I'll select the columns that will be diluted.
I want to dilute from column 2 to 24, so I'll click Select All. I’ll just scroll over to the right to make sure that is the case.
I’ll leave the default order selection, because it is typical.
In step 5, I can verify that the transfer I’ve specified is correct. To do this, I'll click Next transfer repeatedly to see how each step of the operation will occur.
In this case, I’m transferring 30 microliters from column A1 to column A2. Then from column A2 to column A3, and so on.
The concentration of the samples is halved with each transfer.
Next, I can specify aspirate and dispense properties. In this example, I will go ahead and use the default values and selections.
And finally, I get to specify mix parameters. Once again, I will use the default values and settings. Now I’m done with the wizard.
Back in the protocol, I see that all of the necessary serial dilution tasks and parameters are viewable in the Task Parameters area.
I can click the green arrows to step through them or make modifications.
Before I leave the Serial Dilution subprocess, there is one more thing I have to do…
I am going to add the Tips Off task. In the Task Parameters area, I'll select the Tipbox.
Also, I'll clear the check box for Mark tips as used, because I plan to use the same tips for the replication process. Now I’m done with the serial dilution subprocess.