PCR with Taqman Probe on LabBot 3D Printer

This is an example that demonstrates how to configure a LabBot 3D printer to run nucleic acid detection methods using polymerase chain reaction (PCR) with a Taqman probe. To achieve this we developed a special pipetting technique that involved modifying pipette tips to be able to cap tubes. 

The first thing that was done was to create a way to load the samples. Since this uses thermocycling and we do not heat the lid (which is a pipette tip), instead we used mineral oil to keep the sample from evaporating in the tube. Furthermore because of the pipette also functioning as a tube capper, we did some modifications to how the samples were set up for loading. 

Taqman probe PCR labbot 3D printer SARS-CoV-2 detection Covid-19 detection
A flow chart diagram showing the steps involved with aspirating the mineral oil and master mix

After setting up the master mix and mineral oil for aspiration we are ready to go. 

Taqman probe PCR nucleic acid detection labbot deck layout
The layout of LabBot deck that illustrates the objects used in the example Taqman probe PCR based nucleic acid detection assay. The LabBot Robotic Schedular software includes this tool where you can define objects to document your method. 


Step 1  Mineral oil aspiration

The LabBot Robotic Scheduler software makes it easy to assemble macros and save them so that you can point, click and run. Here is how an oil aspiration macro was saved, selected, and run. 

Aspiration mineral oil using LabBot 3D printer for Taqman probe PCR using thermocycling
Aspiration of mineral oil macro using the LabBot Robotic Scheduling Software


Step 2  Master mix aspiration

After the pipettes load the mineral oil, then the master mix is aspirated. They have to be done with these cut down PCR tubes in order to aspirate 10μl of master mix since these pipettes have a gasket membrane that is used for sealing tubes which are done afterward. 

Aspiration master mix using LabBot 3D printer for Taqman probe PCR using thermocycling
Aspiration of 8μl of master mix. The tubes contain 10μl

Step 3 Load tubes using pipette tips

After master mix aspiration the pipette tips are moved to PCR tubes so that they can be loaded and sealed for thermocycling. 

Tube loading using LabBot 3D printer for Taqman probe PCR using thermocycling
Sealing and loading the tubes for thermocycling PCR detection

Step 4 Dispense master mix and mineral oil into tubes

After tube loading, now the pipettes can dispense the master mix and mineral oil that it previously aspirated. 

Taqman probe real time PCR nucleic acid detection SARS-CoV-2 COVID-19 detection dispense into tubes
Dispense master mix and mineral oil into tubes for thermocycling PCR detection


Step 5 Thermocycle and imaging reaction

Now the samples and be incubated and it is possible to image after every cycle or every so many cycles. The macro does the imaging after 5 cycles (95C denaturation for 30 seconds and 65C extension for 2 minutes). 

Thermocycling and imaging for monitoring PCR reaction on LabBot 3D print
Thermocycling and imaging to visualization nucleic acid amplification. For this Taqman assay (95C for 30 seconds and 65C for 2 minutes)

Step 6 Remove the tubes

After the reaction is finished, the system includes a technique for removing the tubes. Then the pipettes can be washed and on to the next set of samples. 

Pipette tip and tube removal tool for LabBot and other 3D printers. Removing Taqman probe PCR tubes
Tube removal technique. After the reaction is finished the tubes are removed using this LabBot removal tool

Example data

When visualizing the fluorescence (using a blue LED and amber color filter in front of a camera). The Taqman probe oligonucleotide DNA sequence changes color from red to green when it binds to the target sequence. So the approach is to quantify the change of color of the course of the thermocycling process. 

This is an example that shows how a 3D printer (LabBot) can be configured to run PCR reactions for nucleic acid detection. 3D printer LabBot real-time PCR Taqman probe nucleic acid detection
Comparison between a template and no template is plotted to demonstrate how the PCR amplified nucleic acid signal can be detected using a Taqman probe.