Growing Bacteriophage M13 in Liquid Culture
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Published on: August 17, 2017
Abstract:
Stocks of bacteriophage M13 are usually grown in liquid culture. The infected bacteria do not lyse but, instead, grow at a slower than normal rate to form a dilute suspension. The inoculum of bacteriophage is almost always a freshly picked plaque or a suspension of bacteriophage particles obtained from a single plaque, as described here. Infected cells contain up to 200 copies of double-stranded, replicative-form DNA and extrude several hundred bacteriophage particles per generation. Thus, a 1-mL culture of infected cells can produce enough double-stranded viral DNA (1-2 mg) for restriction mapping and recovery of cloned DNA inserts and sufficient single-stranded DNA (∼5-10 mg) for site-directed mutagenesis, DNA sequencing, or synthesis of radiolabeled probes. The titer of bacteriophages in the supernatant from infected cells is so high (∼1012 pfu/mL) that a small aliquot serves as a permanent stock of the starting plaque.
Insights
Bacteriophage M13 stocks are grown in liquid culture, yielding high quantities of double-stranded and single-stranded viral DNA. This method provides ample DNA for various molecular biology applications, including sequencing and mutagenesis.
Area of Science:
- Molecular Biology
- Virology
- Biotechnology
Background:
- Bacteriophage M13 is a filamentous phage commonly used in molecular biology.
- Traditional methods for M13 stock propagation involve liquid cultures.
- Infected bacterial cells do not lyse but exhibit reduced growth rates.
Purpose of the Study:
- To describe a method for efficient bacteriophage M13 stock preparation.
- To highlight the yield of viral DNA from infected bacterial cultures.
- To emphasize the utility of M13-derived DNA for molecular biology techniques.
Main Methods:
- Culturing bacteriophage M13 in liquid media with host bacteria.
- Harvesting bacteriophage particles and DNA from the culture supernatant.
- Quantifying double-stranded replicative-form DNA and single-stranded viral DNA.
Main Results:
- A 1-mL culture yields 1-2 mg of double-stranded M13 DNA and 5-10 mg of single-stranded M13 DNA.
- Supernatant titers reach approximately 10^12 plaque-forming units/mL.
- Infected cells produce hundreds of phage particles per generation.
Conclusions:
- This method provides a reliable and high-yield source of M13 viral DNA.
- The produced DNA is suitable for restriction mapping, DNA sequencing, and site-directed mutagenesis.
- A small aliquot of the phage stock can serve as a permanent source for future experiments.
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