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Melissa Martinez

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

Introduction 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
Molecular Genetics

Cloning into Bacteriophage M13 Vectors

Introduction and Principle 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
Molecular Genetics

Generation of Single-Stranded DNA with Phagemid Vectors

Introduction 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.
Molecular Genetics

Hybridization based Screening of Bacteriophage DNA on the filters

Introduction 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
Laboratory Techniques

Large Scale Extraction of Bacteriophage λ DNA Using Proteinase K

Introduction and Principles 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: phenol and ultimately removal of CsCl. The
Laboratory Techniques

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 delivery of Cas9 from Streptococcus pyogenes coupled with synthetic and
Laboratory Techniques

Purification of Bacteriophage λ Arms using Sucrose Density Gradients

Introduction 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 methods are used to purify bacteriophage λ arms: Sucrose
Laboratory Techniques

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

Introduction 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 type of vector with EcoRI and BamHI generates left
Nucleic Acid Quantification Protocols
Laboratory Techniques

Alkaline Phosphatase Treatment of Bacteriophage λ Vector DNA

Introduction Alkaline phosphatase treatment is used to suppress the background of non-recombinants while performing restriction digestion with insertion vectors containing a single cloning site or polyclonal site using a single restriction enzyme. In this method, ligated DNA is first subjected to phenol: chloroform extraction and standard ethanol precipitation, respectively. It is then treated with restriction enzymes. The digested vector DNA