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