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Molecular Genetics

Molecular Genetics

Bacteriophage λ DNA Purification from Liquid Cultures

In the past, bacteriophage λ DNA purification was largely achieved through procedures that involved Cesium chloride gradients. However, these methods had a low recovery rate and a high chance of the presence of contaminants. Modern bacteriophage λ DNA purification methods give high yield and provide DNA free from contamination (Pickard, 2009). We can also purify bacteriophage λ DNA from liquid

Molecular Genetics

Bacteriophage λ DNA Purification from Plate Lysates

Genomic DNA or cDNA clones are analyzed in a simple procedure that begins with the restriction digestion of phage DNA mini preparations. The digested DNA is then assessed by agarose gel electrophoresis. For the rapid analysis, one can purify bacteriophage DNA from plate lysates as well as liquid cultures. This method explains the purification of phage DNA from plate lysates.

Molecular Genetics

Cloning into Bacteriophage M13 Vectors

Insertion of foreign DNA into M13 cloning vectors is a bit tricky and includes the following steps: Ligating double-stranded DNA segments with cohesive termini into compatible sites in M13 double-stranded RF DNA. Transforming ligation products into competent male E. coli cells (plated in top agar supplemented with X-gal and IPTG.  Picking and propagating recombinant (white) and non-recombinant (blue) M13 phages

Molecular Genetics

Generation of Single-Stranded DNA with Phagemid Vectors

The vectors derived from filamentous phages containing a plasmid’s origin of replication are called phagemids (Qi et al., 2012). Phagemids comprise typical high-copy-number plasmids equipped with a major intergenic region (508bp in length) of a filamentous phage. This region does not encode any proteins; however, it comprises all the cis-acting sequences required to initiate and terminate viral DNA synthesis. These

Molecular Genetics

Hybridization based Screening of Bacteriophage DNA on the filters

Filters carrying immobilized bacteriophage DNA are labeled with 32P-labelled probes to screen by hybridization in situ. In this procedure, the filters are first submerged in a prehybridization solution (containing blocking solution) to reduce non-specific probe absorption. The filters are then incubated with a denatured radioactive probe for hybridization. Wash the filters to remove the non-specifically attached probe. Get the image