Melissa Martinez

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:…

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…

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…

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…

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…