E. Coli Top10 Reaction Lab Report
Material and methods
Hosts , plasmids and chemicals
E. coli TOP10 strain was used for cloning and proliferation. E.coli BL21 DE3 was used as expression host cell. The pET 28a(+) plasmid was employed for gene expression. All chemicals were obtained from Merck Company (Germany).
Codon optimization and gene synthesis
Sequence encoding V-domain was obtained from Swiss-port, Uniprot KB and National Center for Biotechnology Information (NCBI) databases. 6x His-tag sequence was placed at N-terminal followed by termination codon (TAA). NcoI and BamHI restriction sites were added at C-terminal and XhoI at N-terminal. Codon optimization was done for Expression in E. coli by the genescript Company (860 Centennial Ave., Piscataway, NJ 08854, USA). The optimized sequence was synthesized and inserted in to the pBSK (+) vector (Biomatik, Canada). Simple pBSK(+) is highly recommended for standard cloning purpose. …show more content…
After plasmid extraction (Fermentas, Lithuania), a double digestion was done with NcoI and XhoI (Fermentas, Lithuania) enzymes. The expression vector pET28a was also digested with NcoI and XhoI. The double digestion was carried out at 37ºC, digested fragments were analyzed by agarose gel electrophoresis and the products were purified (Fermentas, Extraction of DNA from the gel kit, Lithuania).
The digested products, pET28a expression vector and V-Domain fragments, both were ligated by T4 DNA ligase, with the ration of 1:6 of vector and V-domain gene, and incubated first at 16 ºC for 4 h then at 4 ºC for
Hosts , plasmids and chemicals
E. coli TOP10 strain was used for cloning and proliferation. E.coli BL21 DE3 was used as expression host cell. The pET 28a(+) plasmid was employed for gene expression. All chemicals were obtained from Merck Company (Germany).
Codon optimization and gene synthesis
Sequence encoding V-domain was obtained from Swiss-port, Uniprot KB and National Center for Biotechnology Information (NCBI) databases. 6x His-tag sequence was placed at N-terminal followed by termination codon (TAA). NcoI and BamHI restriction sites were added at C-terminal and XhoI at N-terminal. Codon optimization was done for Expression in E. coli by the genescript Company (860 Centennial Ave., Piscataway, NJ 08854, USA). The optimized sequence was synthesized and inserted in to the pBSK (+) vector (Biomatik, Canada). Simple pBSK(+) is highly recommended for standard cloning purpose. …show more content…
After plasmid extraction (Fermentas, Lithuania), a double digestion was done with NcoI and XhoI (Fermentas, Lithuania) enzymes. The expression vector pET28a was also digested with NcoI and XhoI. The double digestion was carried out at 37ºC, digested fragments were analyzed by agarose gel electrophoresis and the products were purified (Fermentas, Extraction of DNA from the gel kit, Lithuania).
The digested products, pET28a expression vector and V-Domain fragments, both were ligated by T4 DNA ligase, with the ration of 1:6 of vector and V-domain gene, and incubated first at 16 ºC for 4 h then at 4 ºC for