A home-made T/A vector for simplification of cloning DNA fragments obtained from PCR

• Main Authors: Nahid Masoudi, Nemat Sokhandan Bashir
• Format: Article
• Language: fas
• Published: Shahid Bahonar University of Kerman 2013-08-01
• Series: مجله بیوتکنولوژی کشاورزی
• Subjects: t/a vector; soybean mosaic virus; preparation of vector; ptz75r/t
• Online Access: https://jab.uk.ac.ir/article_1207_88b20f94e57e151adf0ac956e5bcce02.pdf

Today, genetic engineering is an indispensable component of modern biological research and DNA cloning, as one of the most important applications of this technology, has a wide-range application. Plasmids are the most commonly used vectors that provide replication of a desired gene usually in a bacterial host. T/A cloning vectors are one type of plasmids which facilitate cloning of a DNA fragment provided that the DNA is amplified by <em>Taq</em> DNA polymerase through polymerase chain reaction (PCR). In cases where the resources are limited, purchasing commercial T/A cloning kits may be hardly possible. So, availability of a home-made T/A vector with a good performance in the laboratory would be important. In this study, after preparation of competent <em>Echerchia coli</em> cells, the circular plasmid pTZ57R (without insert) was transformed into the cells. After plasmid extraction by alkaline lysis method, the plasmid was cut with <em>Eco</em>RV to make it linear. Then, the enzyme was inactivated and thymine nucleotide was added to the free 5‘ ends of the linear plasmid. The efficiency of the vector was demonstrated during ligation reactions and subsequent transformations. In addition, a segment of potyvirus genome that was amplified by a pair of universal primers was inserted into the T/A vector and subjected to sequencing. Comparison of the sequencing data with that of the counterpart regions available in GenBank has ended in identification of the virus as <em>Soybean</em><em> mosaic virus</em>.