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Quantitative Polymerase Chain Reaction to Enumerate Bacteriophages
Overview
In this video, we discuss the quantitative polymerase chain reaction, qPCR, to enumerate T7 bacteriophages through DNA quantification. The fluorescent dye in the PCR mixture binds with the newly synthesized DNA strands and emits fluorescence, which is measured.
Protocol
1. Prepare qPCR reactions
NOTE: One PCR reaction is for one sample. Each PCR reaction contains 5 µL of qPCR master mix (see materials), 1 µL of 5 µM primer pair mix, 2 µL of H2O, and 2 µL of heat-treated T7 phage sample. For multiple PCR reactions, all the reagents except the phage sample are premixed in one 1.5 mL tube. The volume of each reagent in the premix depends on the number of phage samples for qPCR.
- Prepare the qPCR pre.......
Representative Results
Figure 1: Phage sample treatment, qPCR reaction preparation, and qPCR run. DNase I pretreated T7 phage samples were heated at 100° C for 15 min to release the T7 DNA from intact phage particles (A); the qPCR mixture was prepared as described in the protocol. All the preparations were done on ice (A); qPCR equipment and compatible software (B.......
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Source: Peng, X., et al., Quantitative PCR of T7 Bacteriophage from Biopanning. J. Vis. Exp. (2018)
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