Quantitative Polymerase Chain Reaction to Enumerate Bacteriophages
Transcrição
Prepare the qPCR premix for 10 biopanned phage samples of unknown concentration and 7 samples of standard concentrations in triplicate, resulting in a premix for 51 samples containing 255 microliters of qPCR Master Mix, 51 microliters of primers, and 102 microliters of water.
Aliquot 8 microliters of the prepared PCR mix into 51 wells of a 96-well qPCR plate. Next, add two microliters of heat-treated T7 phage samples to each of these wells, dispensing it below the surface of the qPCR premix. Using adhesive film, seal the plate.
Wrap the plate in aluminum foil to minimize fluorescence photobleaching, and then, proceed to run the plate on the qPCR equipment. Set up the cycling conditions and melt curve settings as outlined in the text protocol.
