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 […]

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 […]

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 the “preparation of λ arms.” Restriction endonucleases are used to separate the two arms of phage DNA from the central stuffer fragment. The following methods are […]

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 […]

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 […]

The ?-radiation and its effects on chromosomes

Introduction and Historical Perspectives Radiations have been extensively used in medical devices to look into the inner workings of the human body. Wilhelm Roentgen brought a revolution in the medical field by first developing an X-ray machine in 1895 after which, the first radiology department was set up. After the discovery of X-ray, various scientists […]