The overall goal of this procedure is to demonstrate how the principle of PCR Thermocycling by thermal convection can be applied to run an actual reaction. This is accomplished by first introducing the idea of how natural convection thermocycling works, along with the basic design principles. The second step of the procedure is to show how to prepare the reactor cartridge and load the reagents inside.
The third step of the procedure is to assemble the reactor cartridge into the heater unit and run the reaction. The final step of the procedure is to remove the reagents after the reaction and check the results with gel electrophoresis. Ultimately, results can be obtained that show Rapid DNA replication through natural convection PCR Thermocycling.
Hi, I'm Radha OU from the UGAs lab at Texas a and m University. Today we will show you a procedure for performing rapid PCR Thermocycling using natural convection like the lava lamp. We use this procedure in a laboratory to study the design and optimization of thermocycling devices that harness this principle.
So let's get started. This protocol begins with the design and construction of a simple convective thermocycling device. The device used for this demonstration consists of interchangeable plastic reaction chamber cartridges that are sandwiched between two aluminum plates whose temperatures are independently controlled.
Cylindrical reactor wells were embedded by machining arrays of holes in polycarbonate blocks with different combinations of whole diameter and plastic sheet thickness employed to achieve the desired aspect ratios. Optimal design involves choosing the correct reactor geometry that will generate a circulatory flow capable of transporting reagents through the key temperatures involved in the PCR process. The geometric parameters that can be varied in the cylindrical reactors considered Here are the height and diameter or equivalently the aspect ratio.
The bottom surface of the reactor is heated using an aluminum block containing cartridge heaters interfaced with a microprocessor driven temperature controller. The temperature at the reactor's upper surface is regulated using an aluminum block connected to a recirculating water bath. The entire assembly is clamped together using nylon screws to limit thermal conduction between the opposing aluminum blocks.
Prepare each 100 mic release of PCR reaction by mixing 10 times buffer solution 25 millimolar magnesium chloride, two DTPs distilled water, the beta actin probe forward primer and reverse primer human genomic template, DNA and K-O-D-D-N-A polymerase. Before loading reagents, seal the bottom surface of the PCR chamber using a thin sheet of aluminum tape rinse the reactor wells with a 10 milligram per milliliter aqueous solution of bovine serum albumin followed by rain X anti-fog. Finally, pipette reagents into the reactor wells using gel loading tips and seal the upper surface with FEP Teflon tape.
Before beginning the reaction preheat both aluminum blocks to the desired upper and lower surface temperatures. Sandwich the plastic reactor loaded with PCR reagents between the aluminum heating blocks and quickly clamp the assembly together using nylon screws. After the reaction has proceeded for the desired time, switch off the heaters and place the lower heated surface of the device on top of a chilled metal block to rapidly cool it and stop the convective flow.
Remove the plastic reactor and pipette the products outta the wells for subsequent analysis. Prepare a 2%arose gel by heating 10 grams of agros with 500 milliliters of one times buffer on a stirring hot plate until the solution becomes clear. Load the A rose gel into the casting tree and insert the comb.
Let the gel set for approximately 30 minutes. Remove the comb and add one times TAE buffer until the gel is submerged. Prepare fluorescently stained DNA samples with two microliters of 100 times cyber green.
One solution two microliters of DNA sample, two microliters of six times orange loading dye and four microliters of TAE buffer. Load a 100 base pair DNA ladder sizing marker and the DNA samples into the wells of the gel. Separate the DNA by running the gel at 60 volts for one hour.
Remove the gel and photograph it under UV light to obtain the result. 3D flow fields inside convective PCR reactors were investigated over a range of different aspect ratios using computational fluid dynamics or CFD. This data revealed that complex patterns can arise and that a subset of these complex flow fields can significantly accelerate the reaction.
These geometric effects can be clearly seen by comparing the flow fields in reactors at high and low aspect ratios with a high aspect ratio. Fluid elements are affected along trajectories, tracing out paths that are essentially closed loops and are locked in to follow the same paths for long periods of time. Consequently, there is little opportunity for exchange between flow trajectories that expose reagents to the optimal sequence of thermal conditions for PCR and the much larger ensemble of remaining trajectories that do not contribute to amplification.
The flow field is much different at a smaller aspect ratio. The flow field is more chaotic in that the fluid element trajectories no longer follow closed paths. Although the reactions are subjected to a more complex temperature profile, fluid elements are able to explore a much wider range of trajectories so that more of the reagent volume has an opportunity to experience optimal thermal profiles.
These results suggest that while individual flow trajectories at H over D equals nine may appear to be more favorable for PCR by producing temperature profiles that look similar to those employed in a conventional thermocycler. The chaotic nature of the flow field at HOD equals three ultimately dominates on a global scale by promoting enhanced exchange so that reagents do not become trapped in unfavorable trajectories for very long. To test this hypothesis and determine which reactor geometry was more favorable for PCR, both cylinders were used to perform PCR replication of a 295 base pair target associated with the beta actin gene from a human genomic DNA template amplification of the correct target product was repeatedly achieved in only 10 minutes at H over D equals three.
Conversely, the same reaction required at least 20 minutes before detectable products were observed at H over D equals nine. The reaction specificity is also much greater at H over D equals three where a single PCR product is obtained. While multiple non-specific products are generated at H over D equals nine, where the flow field traps fluid elements in unfavorable thermal trajectories for extended periods of time.
We've just shown you how to run a PCR reaction using a convective flow.Thermocycler. When doing this procedure, it is important to remember to seal the reagents inside the reactor chamber without dropping any air pockets. These would disturb the flow field inside and inhibit the reaction.
So that's it. Thank you for watching and good luck with your experiments.
