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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.

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Before I do that, let’s briefly review what we’ve done so far in the protocol.

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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.

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Now I need to dilute the column serially in the destination plate.

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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.

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The destination plate remains at Bravo deck location 6.

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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 

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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.

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After adding the diluent, I can add the serial dilution tasks.

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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.

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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

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serial dilution. This will be done at the Multidrop device, so I need to add the Multidrop task.

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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.

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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 

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through 24. Then I'll click the next row, and click the browse button that appears to select the desired columns.

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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.

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Then I'll click the A1 well to clear the A1 column.

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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.

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I’ll click OK to go back to the protocol and check that the well-column selection is reflected in the Task Parameters area.

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I’m going to leave the rest of the parameters. And I’m done with this task.

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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.

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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.

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In the Spawn Process Task Parameters area, I'll select Tipbox for Serial Dilution and Replication.

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I don’t need to select the Spawn as subroutine option, because the Tipbox process will not be a subroutine of the Destination process.

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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

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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.

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For detailed information about accessible deck locations for different pipette channel selections, see the VWorks Automation Control User Guide.

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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.

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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.

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So the Destination plate can use that location for the serial dilution tasks.

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When the Tipbox process is spawned, the first thing that should happen is the tipbox will be downstacked from the BenchCel stacker.

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So I'll add the Downstack task in the Tipbox process.

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The tipbox will be coming from BenchCel stacker 5.

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And according to the plan, it should be placed at deck location 7 for the serial dilution and replication tasks.

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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.

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Because the tipbox is also used in the serial dilution tasks, I need to include it in the subprocess.

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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.

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Let’s start to add the tasks in this subprocess.

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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…

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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.

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Let’s press on the tips. I'll add the Tips On task.

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In the Task Parameters area, I'll select the Tipbox.

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I can leave the well selection at A1, because the software will refer to the channel selection in the Set Head Mode task.

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Now I’ll add the Serial Dilution task.

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The serial dilution will be performed in the Destination plate, so I'll select it.

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I'll click Launch serial dilution wizard, which will walk me through the setup process.

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In Step 1, I get to specify how I want the liquid transfer to occur and how much.

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I want the dilution to be based on transfer volume, which is already selected, so I'll move on.

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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.

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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.

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I don’t want to specify other operations, so I’ll leave the No additional operations selection and move on.

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For an explanation of these other options, see the VWorks Automation Control User Guide.

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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.

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Next, I'll select the columns that will be diluted.

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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.

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I’ll leave the default order selection, because it is typical.

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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.

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In this case, I’m transferring 30 microliters from column A1 to column A2. Then from column A2 to column A3, and so on.

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The concentration of the samples is halved with each transfer.

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Next, I can specify aspirate and dispense properties. In this example, I will go ahead and use the default values and selections.

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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.

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Back in the protocol, I see that all of the necessary serial dilution tasks and parameters are viewable in the Task Parameters area.

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I can click the green arrows to step through them or make modifications.

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Before I leave the Serial Dilution subprocess, there is one more thing I have to do…

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I am going to add the Tips Off task. In the Task Parameters area, I'll select the Tipbox.

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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.

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