James was tasked to extract DNA from lambda and M13 phages for use as cloning vectors....

James was tasked to extract DNA from lambda and M13 phages for use as cloning vectors. M13 DNA can exist as single stranded or double stranded (replicative) forms. Lambda DNA can exist as both linear and circular forms.
During the extraction of the lambda DNA, it was observed that the extracellular titre of phages was extremely low due to their lysogenic state. Describe two approaches that could be adopted to overcome this hurdle.

Expert Solution

Traditionally,Three method have been used to quantitate bacteriophase.

1.Plaque counts on agar plates seeded with the bacteria in which the phases can propagate.

2.Dilution method,where bacterial lysis is used as an indicator of phase presence.

3.Measuring the length of time required to lyse a standarized bacterial suspension. Another methods are Transmission electron microscopy after negative staining or by epifluorescence microscopy but they have some limitations.

Recently,two new rapid assays are informed for determining phase concentrations.When extracellular titre of phases was extremely low due to their lysogenic state.

1.A quantitative real-time PCR based approach.This assay correlate DNA counts and number of viable phase particles.In this technique phase-containing specimen is amplified.

2.A Nanoparticle tracking analysis(NTA) - based approach using NanoSight Limited(NS) technology.This approach utilizes laser-illuminated optical microscopy for direct ,real time visualization of nanoparticles in a clear liquid.They are counted in a few seconds or a few minutes.

These techniques are used in determining phase concentrationc

gladiator answered 2 years ago

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