Chip-in-a-Tube-Based Digital PCR for Quantification of Single Nucleotide Variants
Transcripción
Add previously-designed primers, probes, and genomic DNA to a new 8-tube strip, to achieve a total volume of 15 microliters. Pipette up and down to mix.
Add the loading platform onto the chips built in the fresh 8-tube strip, and then, place the tube strip in the auto-loader. Make sure that there is a contact between the chips and the loading platform. Next, place the loading slider in the platform, and use a stopper to hold the slider off of the loader. Pipette 15 microliters of the PCR mixture near the tip of the slider, and then, press the loader button to run the loader for 1 minute.
Remove the tube strip from the loader after the run, and place it in sealing enhancer. Carefully push the slide lid and the edge of the top lid. Run the sealing enhancer for approximately 2 minutes. If sealing is incomplete, indicated by a puddle of liquid, repeat the run for an additional minute.
Add 230 microliters of sealing fluid to the tubes. Place the tube strip in the thermal cycler, and run the PCR as described in the protocol. If there is an uneven distribution of positive partitions, adjust the temperature or the duration of the PCR.
To detect and analyze fluorescence intensity of the PCR products, place the tube strip on the detection jig, and add 6 milliliters of distilled water. Remove any visible air bubbles using a pipette tip.
Load the jig into the detector. In the detection software, select "Fluorescence," "Experiment," and then "Sample/NTC" tabs, and click the "Run" button to start the run. After the complete run, confirm the position plot, histogram, and 2D scatterplot.
To collect the PCR product, remove the sealing fluid from the tube. Add 100 microliters of TE buffer, and vortex vigorously for 30 seconds. Briefly centrifuge the tubes in a tabletop centrifuge, and proceed as described in the text protocol, to finish collecting the PCR product.
