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.
Before I do that, let’s briefly review what we’ve done so far in the protocol.
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.
Then I added tasks to dilute the column serially across the destination plate. Now I want to make two copies of the diluted plate.
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.
The tips used for serial dilution are at deck location 7. I’ll reuse the same tips during the replication process.
The new plates we’ll use for replication are stacked at Bravo deck location 1.
During the plate replication process, the plates for replication will downstack from Bravo deck location 1 and go to location 8.
I’ll use the tips in the tipbox at deck location 7.
OK, let’s add the replication tasks in the protocol.
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
Serial Dilution subprocess to initiate the Replication Plate process.
And I'll select Replication Plate, because that’s the process I want to spawn.
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.
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.
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.
I'll click Add Process…
…then add Scan Stack.
Over here in the Properties area, I'll select Bravo and location 1.
OK, I'll go back to the Main Protocol.
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.
So I'll add a Place Plate in the Replication Plate process, select Bravo, and select 8.
Now I’m going to add the replication subprocess.
I'll add a Bravo Subprocess…
I'll name it Replicate.
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.
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.
So I'll paste another copy of the Replication subprocess in the Tipbox process.
Now I’m ready to add tasks in the replication subprocess.
The first task I’m going to add is the Tips On task… I'll select the Tipbox for Serial Dilution and Replication.
And over to Well selection… I want the entire plate, which is already done.
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.
With the tips on, I can start to aspirate… from the Destination plate.
I want to aspirate 20 microliters. My plan is to dispense 10 in each of the two replication plates.
I’ll use the defaults for the remaining parameters.
I'll add the Dispense task. I want to dispense into the replication plate, so I'll select that.
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.
I need a way to dispense into the second replication plate. I can add three tasks to help me do this.
First, I'll add the Change Instance task.
For Plate to change, I'll select the Replication Plate, because that’s the labware for this task.
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.
The other spawn control selections are explained in detail in the VWorks Automation Control User Guide.
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.
I want to dispense twice, so I'll add the first Loop task just before the Dispense task.
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.
I'll also add the Loop End task after the Dispense task so that only the Dispense task is repeated.
At the end of the replication, I want to remove the tips. So I'll add the Tips Off task.
I want to put the used tips back in the same box, so I select the Tips for Serial Dilution and Replication.
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.
Finally, I want to make sure all of the tips are removed.
Now it’s time to upstack the plates so that they are put away at the end of their processes.
I'll first upstack the Destination plate.
It’s going back to BenchCel stacker 2.
Then, I'll upstack the Source plate.
It’s going back to BenchCel stacker 4.
And I'll upstack the Tipbox for Serial Dilution and Replication.
It’s going back to BenchCel stacker 6.
Lastly, I want to upstack the Replication Plate.
It downstacked from Bravo location 1, and I want to upstack it at Bravo location 4.
Again, I need to add a Scan Stack task in Startup Protocol to cover this Bravo Upstack task.
So I'll go over to the Startup Protocol and set this up for the Bravo device, at location 4.
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.
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
will be finished before liquid-handling tasks for the serial dilution starts, and serial dilution must be finished before the replication can begin.
And with that, my protocol is finished!