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In the Serial Dilution video, I showed you how to include serial dilution tasks in a protocol. Now I’ll show you how to add plate replication 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 previous two videos, 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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Then I added tasks to dilute the column serially across the destination plate. Now I want to make two copies of the diluted plate.

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Let’s also check where things are at the devices. At the end of the serial dilution routine, the destination plate is at Bravo deck location 5.

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The tips used for serial dilution are at deck location 7. I’ll reuse the same tips during the replication process.

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The new plates we’ll use for replication are stacked at Bravo deck location 1.

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During the plate replication process, the plates for replication will downstack from Bravo deck location 1 and go to location 8.

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I’ll use the tips in the tipbox at deck location 7.

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OK, let’s add the replication tasks in the protocol.

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Before I do that, I have to get things ready. Once again, I want to hold back the plates I will use for replication until I’m ready, so I'll add a Spawn Process task after the 

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Serial Dilution subprocess to initiate the Replication Plate process.

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And I'll select Replication Plate, because that’s the process I want to spawn.

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Next, I need to specify where the replication plates will be. Remember that in the Replication plan, the replication plates are stacked at Bravo deck location 1.

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I need to downstack each from location 1 for the replication. So I'll add a Downstack task in the Replication Plate process, and select Bravo deck location 1.

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For each Bravo Downstack and Upstack task, I also have to add a Scan Stack task in the Startup Protocol. So I'm going to go to the Startup Protocol and do that.

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I'll click Add Process…

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…then add Scan Stack.

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Over here in the Properties area, I'll select Bravo and location 1.

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OK, I'll go back to the Main Protocol.

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There is one last thing I have to do. After the replication plate is downstacked, I have to specify where it will go. In the plan, I specified location 8.

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So I'll add a Place Plate in the Replication Plate process, select Bravo, and select 8.

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Now I’m going to add the replication subprocess.

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I'll add a Bravo Subprocess…

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I'll name it Replicate.

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And besides the Destination plate, the Replication plate is also involved in this subprocess. So I'll copy the Replicate subprocess and paste it in the Replicate Plate process.

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Remember that we need some tips for this part of the protocol as well, and that I want to reuse the tips that were used for serial dilution.

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So I'll paste another copy of the Replication subprocess in the Tipbox process.

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Now I’m ready to add tasks in the replication subprocess.

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The first task I’m going to add is the Tips On task… I'll select the Tipbox for Serial Dilution and Replication.

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And over to Well selection… I want the entire plate, which is already done.

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Notice I didn’t add the Set Head Mode task. This is because with each new subprocess, the default channel selection is all channels, which is what I want.

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With the tips on, I can start to aspirate… from the Destination plate.

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I want to aspirate 20 microliters. My plan is to dispense 10 in each of the two replication plates.

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I’ll use the defaults for the remaining parameters.

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I'll add the Dispense task. I want to dispense into the replication plate, so I'll select that.

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And I want to dispense 10 microliters into each plate. So far, I’ve aspirated once and picked up 20 microliters and dispensed once and dropped 10 microliters.

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I need a way to dispense into the second replication plate. I can add three tasks to help me do this.

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First, I'll add the Change Instance task.

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For Plate to change, I'll select the Replication Plate, because that’s the labware for this task.

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Then for Spawn control, I'll select Spawn new plates when task runs, so that every time this task runs, a new replication plate will be used.

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The other spawn control selections are explained in detail in the VWorks Automation Control User Guide.

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Next, I need to add the two Loop tasks. The Loop tasks tell the software to repeat the tasks within the two Loop tasks a specified number of times.

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I want to dispense twice, so I'll add the first Loop task just before the Dispense task.

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For the Number of times to loop, I'll specify 2. I'll leave the other parameter alone, because I don’t need to change tips after every dispense.

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I'll also add the Loop End task after the Dispense task so that only the Dispense task is repeated.

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At the end of the replication, I want to remove the tips. So I'll add the Tips Off task.

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I want to put the used tips back in the same box, so I select the Tips for Serial Dilution and Replication.

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And I’m going to clear the check box for Mark tips as used, because I will reuse them for the next round of replication.

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Finally, I want to make sure all of the tips are removed.

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Now it’s time to upstack the plates so that they are put away at the end of their processes.

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I'll first upstack the Destination plate.

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It’s going back to BenchCel stacker 2.

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Then, I'll upstack the Source plate.

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It’s going back to BenchCel stacker 4.

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And I'll upstack the Tipbox for Serial Dilution and Replication.

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It’s going back to BenchCel stacker 6.

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Lastly, I want to upstack the Replication Plate.

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It downstacked from Bravo location 1, and I want to upstack it at Bravo location 4.

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Again, I need to add a Scan Stack task in Startup Protocol to cover this Bravo Upstack task.

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So I'll go over to the Startup Protocol and set this up for the Bravo device, at location 4.

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Let’s now return to the Main Protocol… With all of the labware now put away, there is one last thing I need to do to the protocol.

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I am going to go to Protocol Options… And clear the check box for Pipette plates in instance order, so that all of the liquid-handling for hit-picking 

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will be finished before liquid-handling tasks for the serial dilution starts, and serial dilution must be finished before the replication can begin.

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And with that, my protocol is finished!

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