摘要
Whenever there is no adequate DNA replication <em>in vitro</em>, there are alternatives strategies to insert a piece of DNA into a convenient replicon such as small plasmids and bacteriophages. These are called vectors or cloning vehicles. These days, there is a high demand to manufacture recombinant and nanobodies which are required in biomedical research and for therapeutic and diagnostic purposes. In order to do so, <em>E. coli</em>, insect cells, or mammalian cells have been used to express and purify protein for nanobody production. This paper explains the experimental trials on the cloning of the Nanobody cDNA mock-up in pHEN6c plasmid vector from the subcultured <em>E. coli</em> sample taken from the lab. The concentration and purity of DNA plasmid were evaluated by UV spectrophotometer and agarose gel electrophoresis following plasmid DNA Purification from <em>E. coli</em> by alkaline lysis. Based on this, the concentration of the isolated pHEN6c plasmid DNA was found 44.7 ng/μl or 0.0447 μg/μl. Whereas, the purity at absorbance (A260/A280) was 0.893/0.501 = 1.78. Moreover, its yield was 2.235 μg. In addition, its transformation efficiency was 21.68 μg/μl. On the other hand, the molecular weight of the Nanobody and vector were 569 and 2717 respectively. Generally, most of the protocols used to clone a fragment of DNA, might not work very well with PCR products.
Whenever there is no adequate DNA replication <em>in vitro</em>, there are alternatives strategies to insert a piece of DNA into a convenient replicon such as small plasmids and bacteriophages. These are called vectors or cloning vehicles. These days, there is a high demand to manufacture recombinant and nanobodies which are required in biomedical research and for therapeutic and diagnostic purposes. In order to do so, <em>E. coli</em>, insect cells, or mammalian cells have been used to express and purify protein for nanobody production. This paper explains the experimental trials on the cloning of the Nanobody cDNA mock-up in pHEN6c plasmid vector from the subcultured <em>E. coli</em> sample taken from the lab. The concentration and purity of DNA plasmid were evaluated by UV spectrophotometer and agarose gel electrophoresis following plasmid DNA Purification from <em>E. coli</em> by alkaline lysis. Based on this, the concentration of the isolated pHEN6c plasmid DNA was found 44.7 ng/μl or 0.0447 μg/μl. Whereas, the purity at absorbance (A260/A280) was 0.893/0.501 = 1.78. Moreover, its yield was 2.235 μg. In addition, its transformation efficiency was 21.68 μg/μl. On the other hand, the molecular weight of the Nanobody and vector were 569 and 2717 respectively. Generally, most of the protocols used to clone a fragment of DNA, might not work very well with PCR products.
作者
Tewodros Fentahun Jember
Tewodros Fentahun Jember(Department of Veterinary Biomedical Sciences, College of Veterinary Medicine and Animal Sciences, University of Gondar, Gondar, Ethiopia)
