HTS Resources: Software engineering, 3D printing, instrumentation design and documentation
Portable DNA Thermocycler
This is a small thermocycler used for running PCR reactions. It includes a tool for visualizing fluorescence too. It can be used for running CDC approved Taqman tests for SARS-CoV-2.
The system includes:
Milled thermoblock and case for holding PCR tubes
Illuminated tube holder for fluorescence imaging using something like a cell phone camera
Arduino microcontroller shield for controlling heater, Peltier cooling system, and reading a temperature sensor
Simple open-source command-line python based software for thermocycling and turning on blue LED (Works on Windows, Mac and Linux)
CAD view of the thermocycler assembly. It has an imaging station for visualizing fluorescence using something like a cell phone camera. When the Taqman probe binds to the amplified sequence the color change goes from red to green.Picture of what the thermocycler device looks like. Its made using 3D printer parts (ie., heaters, thermistor, electronics and Arduino based firmware)
Portable thermocycler and imaging station for cell phones
The LED excites the fluorochrome and gets you to see if the target sequence is amplified
The Green channel was plotted to compare. Heat conditions:
95C 15 seconds
60C 30 seconds
40 cycles
Samples
beta-actin – PCR with Taqman probe
blank – water control with master mix
Note: after 30 cycles signal was visible
Example of Taqman PCR using a thermocycler made using parts of a 3D printer extruderExample of what the amplfied signal looks like when running a Taqman probe based PCR. When the probe binds to the amplified target sequence the signal shifts from red to green
Pipetting setup for decentralized Taqman probe PCR
The system shown consists of a reaction master mix tube that can contain the sample, mineral oil tube (for keeping the reaction mix at bottom of PCR tube during thermocycling), pipettor tool, wide-bore pipette for aspirating and dispensing mineral oil, and a small volume pipette for aspirating and dispensing the reaction master mix. Also shown are PCR tubes that fit in the PCRmon thermocycler. The mineral oil is layered on top of the aspirated reaction mix.The pipettor is able to securely attach to different types of pipette tips. By squeezing the top part of the silicone tubing, it is possible to aspirate and dispense very small volumes. To consistently aspirate the same volume, a marking on the pipette tip can be drawn as a reference.In order to transfer the reaction from the source well into the PCR test tube, you need to aspirate then dispense the sample. This example demonstrates how it is easy to accurately aspirate microliter volumes of reaction mix (in this action the volume aspirated is 10µl). In order to aspirate, first, you squeeze the tubing at the top of the pipette using your index finger and thumb in order to reduce the volume in the pipette, then you move the pipette into the tube containing the reaction mix that you want to aspirate and carefully reduce the squeezing pressure in order to create suction to aspirate the volume enough to reach the marketing. Then you move the pipette tip to the empty PCR tube and dispense the volume.Because the PCRmon thermal block does not have a heated lid, mineral oil is needed in order to keep the volume of the reaction mix at the bottom of the PCR tube during thermocycling. The smaller pipettes used for aspirating the reaction mix have too small of an orifice for this relatively viscous mineral oil so a larger bore pipette tip is better for this. The volume of the mineral oil added to the reaction mix is not as critical, you can aspirate the total amount of volume (just squeeze and release entirely when the pipette tip is in the mineral oil), this produces around 30μl of mineral oil in the tube.