Digital Droplet Polymerase Chain Reaction for Indel Mutation Detection in Target Genes
Transcrição
To begin with, prepare 25 microliters of the digital droplet PCR or ddPCR sample mix by adding ddPCR super mix, forward and reverse primers, HEX or FAM probes, DNA, and water in the proportions described in the text. Then, thoroughly mix the reaction by vortexing.
Using a 50-microliter multichannel pipette, load 20 microliters of the ddPCR sample mix into the middle row of the cartridge. Then, use a 200-microliter multichannel pipette to load 70 microliters of the oil into the bottom row without generating bubbles throughout the pipetting.
Place the gasket, touching only the edges, avoiding the center concaved area, and then, place the plate securely in the droplet generator, and close the cover to start the run.
To transfer the immersion mix from the top row of the cartridge into the 96-well plate, use a multichannel pipette and draw 40 microliters of liquid sample for 3 to 5 seconds at an angle of 30 to 45 degrees. Then, expel the mixture into the well slowly for over 3 seconds at a 45-degree angle, allowing it to drop down from the side, and then, go to the second stop of the pipette to expel the liquid completely. Using foil heat seals, seal the plates for 5 seconds at 180 degrees Celsius.
Place the sealed plate into the thermocycler and set the PCR conditions for NHEJ Drop-Off guidelines by setting initial denaturation at 95 degrees Celsius for 10 minutes. Then, set 40 cycles of 94 degrees Celsius for 30 seconds to denature, 55 degrees Celsius for 1 minute to anneal, and 60 degrees Celsius for 2 minutes to extend.
After 40 cycles, set hold at 98 degrees Celsius for 10 minutes, and final hold at 4 degrees Celsius. Use a ramp rate of 2 degrees Celsius per second for all steps. The annealing temperature will vary according to the probes or primer used. Place the plate securely in the droplet reader with the A1 labeled well at the top left.
Open the program and set up the plate by designating FAM as the known reference channel and HEX as the unknown one for each well. Then, change the name for each sample. Run the droplet reading experiment as direct quantification while saving the template.
