China
Africa
Explore chapters and articles related to this topic
Genomics and Bionanotechnology
Published in Anil Kumar Anal, Bionanotechnology, 2018
DNA cloning can be either single step or series of steps united with cloning strategy. The multistep approach includes (Tolmachov 2011): Insertion of multiple cloning site also known as polylinker to facilitate subsequent cloningSubcloning of the insert inside multiple cloning site to change restriction sitesSubcloning to change antibiotic selection marker on the recombinant plasmidStepwise insertion of individual genetic elementsTwo-step pop-in-pop-out strategy where insert is transferred from one to another vector
Protein Expression Methods
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
Typically, protein expression vectors include tags for purification and immunohistological identification of the heterologously expressed protein. In most cases, tags are appended to the N-terminus or C-terminus of the protein during subcloning into the protein expression vector. If C-terminal tags are to be appended to the target protein, then the protein must be inserted into the vector without a stop codon. When the vector contains N-terminal tags, care must be taken to insert the gene in frame with the start codon before the N-terminal tags. If a vector contains C-terminal tags that one does not want to append to the protein of interest, it is typically sufficient to leave the native stop codon in place during the subcloning. However, if the vector contains N-terminal tags that one does not want to append to the protein of interest, these tags must be removed from the vector during subcloning. While tags are typically inserted at only either the N-terminus or the C-terminus of the protein, it is possible to insert tags for purification or immunohistological identification into the core of the protein. This strategy has been successfully applied to purification tags inserted into the extracellular loops of multipass transmembrane proteins.
Laboratory tutorials
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
Choice of primers: A number of considerations go into designing primers. Primers should not be self-complementary or complementary to each other, especially at their 3′ ends, to avoid primer dimers from forming. Keep the guanine and cytosine (G + C) content between 40% and 60%. Avoid long stretches of G + C, since they may form secondary structures. Addition of restriction enzyme sites to the 5′ end of the primers serves as useful vehicles for subsequent subcloning. Primer concentrations should be in excess of the template throughout the cycling. Typically, the primers are used over a 0.1–1.0 µM range. Lower concentrations may reduce artifacts and formation of primer dimers.
Nonviral gene delivery using PAMAM dendrimer conjugated with the nuclear localization signal peptide derived from human papillomavirus type 11 E2 protein
Published in Journal of Biomaterials Science, Polymer Edition, 2021
Jeil Lee, Yong-Eun Kwon, Jaegi Kim, Dong Woon Kim, Hwanuk Guim, Jehyeong Yeon, Jin-Cheol Kim, Joon Sig Choi
The luciferase expression plasmid DNA (pCN-Luci) was constructed by subcloning the Photinus pyralis luciferase cDNA with a nuclear localization signal derived from SV40 large T antigen as reported previously [17]. Briefly, pCN-Luci was transformed into E. coli TOP10 competent cells and cultured in large quantities. Then, pCN-Luci was isolated from cultured E. coli cells using the NucleoBond Xtra Midi Plus kit (Macherey-Nagel, Dueren, Germany). Finally, pCN-Luci was precipitated, washed in 2-propanol, and resuspended in distilled water. The plasmid DNA was stored at −20°С until further use.
Increased removal of cadmium by Chlamydomonas reinhardtii modified with a synthetic gene for γ-glutamylcysteine synthetase
Published in International Journal of Phytoremediation, 2020
René Piña-Olavide, Luz M. T. Paz-Maldonado, M. Catalina Alfaro-De La Torre, Mariano J. García-Soto, Angélica E. Ramírez-Rodríguez, Sergio Rosales-Mendoza, Bernardo Bañuelos-Hernández, Ramón Fernando García De la-Cruz
The construct pRPO2 was generated after subcloning the sequence for gshA into the vector p463 (pre-digested with NcoI) at a 1:10 vector-to-insert ratio (Figure 1). To purify DNA the set used was a QIAEX II gel extraction kit (Qiagen GmbH., Hilden, Germany). From selected positive clones based on restriction profiles with EcoRI and SpeI, the expected fragments at 7565 bp and 2493 bp were obtained and analyzed via PCR using forward 5′-CATGGGAGGAGGTCCACCTGTATGATTCCAGA-3′ (1 F) and reverse 5′-ACATGTTCATGACATACAGGTGGACCTCCTCTT-3′ (1 R) with an annealing temperature of 63.1 °C.