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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
Nucleic Acid Quantification Protocols
Laboratory Techniques

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 such as Henri Becquerel, Marie,