Whenever there is no adequate DNA replication in vitro, 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, E. coli, 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 E. coli 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 E. coli 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.
References
[1]
Russel, P.J. (2005) Genetics: A Molecular Approach. Pearson Education, San Francisco.
[2]
Shah, J.M., Ramakrishnan, A.M., Singh, A.K., Ramachandran, S., Unniyampurath, U., Jayshanka, A., Balasundaram, N., Dhanapal1, S., Hyde4 G. and Baskar, R. (2015) Suppression of Different Classes of Somaticmutations in Arabidopsis by Virgene-Expressing Agrobacterium Strains. BMC Plant Biology, 15, 210.
https://doi.org/10.1186/s12870-015-0595-1
[3]
Rusche, J.R. and Howard-Flanders, P. (1985) Hexamine Cobalt Chloride Promotes Intermolecular Ligation of Blunt end DNA Fragments by T4 DNA Ligase. Nucleic Acids Research, 13, 1997-2008. https://doi.org/10.1093/nar/13.6.1997
[4]
Stemmer, W.P., Crameri, A., Ha, K.D., Brennan, T.M. and Heyneker, H.L. (1995) Single-Step Assembly of a Gene and Entire Plasmid from Large Numbers of Oligodeoxyribonucleotides. Gene, 164, 49-53.
https://doi.org/10.1016/0378-1119(95)00511-4
[5]
Viovy, J.-L. (2000) Electrophoresis of DNA and other Polyelectrolytes: Physical Mechanisms. Reviews of Modern Physics, 72, 813-872.
https://doi.org/10.1103/RevModPhys.72.813
[6]
Sambrook, J. and Russel, D.W. (2001) Molecular Cloning: A Laboratory Manual. 3rd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
[7]
Sambroock, J., Fritsche, E.F. and Manniatis, T. (2001) Molecular Cloning: A Laboratory Manual. 3rd Edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
[8]
Steven, A., Baumeister, W., Johnson, L.N. and Perham, R.N. (2016) Molecular Biology of Assemblies and Machines. 1st Edition. Bioscience, New York.
https://doi.org/10.1201/9780429258763
[9]
Primrose, S.B. and Twyman, R.M. (2007) Principles of Gene Manipulation and Genomics. 7th Edition. Blackwell Publishing, Oxford.
[10]
Watson, D.J., Caudy, A.A., Meyers, and Witkowski, R.M. (2007) Recombinant DNA Genes and Genomes-A Short Course. 3rd Edition. W. H. Freeman and Company, Cold Spring Harbor, New York.
[11]
Lodish, H., Berk, A., Zipursky, S.L., Matsudaira, P., Baltimore, D. and Darnell, J. (2000) Molecular Cell Biology. 4th Edition. W. H. Freeman, New York.
https://www.ncbi.nlm.nih.gov/books/NBK21475/
[12]
Brown, T.A. (2008) Gene Cloning & DNA Analysis. An Introduction. 5th Edition. Blackwell Publishing, Oxford.
[13]
Birnboim, H.C. (1983) A Rapid Alkaline Extraction Method for the Isolation of Plasmid DNA. Methods in Enzymology, 100, 243-255.
https://doi.org/10.1016/0076-6879(83)00059-2
[14]
Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring, NY.
[15]
Hanahan, D. and Bloom, F.R. (1996) Mechanisms of DNA Transformation. In: Neidhardt, F.C., Curtiss III, R.C., Ingraham, J.L., Lin, E.C.C., Low, K.B., Magasanik, B., Reznikoff, W.S., Riley, M., Schaechter, M. and Umbarger, H.E., Eds., Escherichia coli and Salmonella Cellular and Molecular Biology, ASM Press, Washington, 2449-2459.
[16]
Tang, X., Nakata, Y., Li, H.O., Zhang, M., Gao, H., Fujita, A., Sakatsume, O., Ohta, T. and Yokoyama, K. (1994) The Optimization of Preparations of Competent Cells for Transformation of E coli. Nucleic Acids Research, 22, 2857-2858.
https://doi.org/10.1093/nar/22.14.2857
[17]
Berg, J.M., Tymoczko, J.L. and Stryer, L. (2002) Biochemistry. 5th Edition. W. H. Freeman and Company, New York.
