Melissa Martinez, Author at Conduct Science

Molecular Genetics Science

June 14, 2021

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,…

Melissa Martinez

Molecular Genetics Science

June 14, 2021

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…

Melissa Martinez

Molecular Genetics Science

June 14, 2021

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. …

Melissa Martinez

Molecular Genetics Science

June 14, 2021

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…

Melissa Martinez

Molecular Genetics Science

June 14, 2021

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…

Melissa Martinez

Laboratory Techniques Science

March 29, 2021

Large Scale Extraction of Bacteriophage λ DNA Using Proteinase K

Large-scale extraction of phage DNA involves digestion of viral coat proteins using a strong protease such as proteinase K followed by extraction with chloroform. This method is suitable for small-scale preparations as well. In the experiment, dialysis of bacteriophage suspension is followed by the extraction of phage particles with chloroform:…

Melissa Martinez

Laboratory Techniques Science

March 29, 2021

Electroporation-based CRISPR Cas9 delivery

CRISPR Cas9 is a potent and easy-to-use tool for gene editing, but the delivery of the CRISPR Cas9 system has been a challenge since it was developed. Several methods have been developed to effectively transport components of the CRISPR Cas9 system into the cells. The protocol below describes the electroporation-based…

Melissa Martinez

Laboratory Techniques Science

March 29, 2021

Purification of Bacteriophage λ Arms using Sucrose Density Gradients

Replacement vectors or substitution vectors are prepared by removing the central filler segment of phage DNA to accommodate the fragments of foreign DNA. This procedure is known as "preparation of λ arms." Restriction endonucleases are used to separate the two arms of phage DNA from the central stuffer fragment. Following…

Melissa Martinez

Laboratory Techniques Science

March 28, 2021

Preparation of Bacteriophage λ DNA Cleaved with two Restriction Enzymes to be used as a Cloning Vector

At each end of the central stuffer fragment, a series of restriction sites arranged in opposite directions are present in replacement vectors. For instance, in EMBL3A, the order of restriction site in the left polyclonal site is SalI-BamHI-EcoRI, and that of the right polyclonal site is EcoRI-BamHI-SalI. Digestion of this…

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Melissa Martinez