DNA plasmids encoding the catalytic domain containing amino acid residues 111–340 of the original SB transposase sequence35 (link) or its hyperactive SB100X version33 (link) were ordered from GenScript USA (Piscataway, NJ). Plasmids were transformed into chemically competent BL21-A1 E. coli cells. Cells were grown in LB medium at 30oC in the presence of ampicillin at the concentration of 0.01 mg/mL until OD600 reached 0.6–0.8. Protein expression was induced by adding 1 mM IPTG and 0.2% L-arabinose for 4 hrs. Cells were collected by centrifugation and lysed in 50 mM TRIS and 500 mM NaCl lysis buffer at pH 8.0 by sonication. Soluble extract containing catalytic domain of SB transposase was prepared by centrifugation of cell lysate at 20,000 g for 1.5 hrs followed by overnight resuspension of pellet, and another centrifugation of cell lysate at 15,000g for 1hr. The protein was purified using Ni-NTA affinity resin (ClonTech, Mountain View, CA) and refolded by gradual decrease of urea concentration. The final sample was prepared in 25 mM TRIS buffer at pH 8.0. The presence and purity of proteins were monitored by SDS-page gel electrophoresis.
Dna plasmids
Manufactured by GenScript
Sourced in United States, Hong Kong
DNA plasmids are circular, double-stranded DNA molecules that can replicate independently within a host cell. They are commonly used as vectors in molecular biology and genetic engineering applications to facilitate the cloning, expression, and manipulation of genes.
Automatically generated - may contain errors
2 protocols using dna plasmids
1
Purification of Hyperactive SB Transposase
2
High-Yield Refolding and Purification of HLA Complexes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein expression, refold and purification were performed as described previously.
26 (link) In brief, DNA plasmids (GenScript, Hong Kong, China) encoding the HLA‐B*35:01, HLA‐B*35:03 and HLA‐B*35:05 heavy chain (1–275 amino acid) and β2‐microglobulin were transformed into the BL21 strain of Escherichia coli cells. Both proteins were expressed separately as inclusion bodies and purified from the transformed E. coli cells. Soluble pHLA complexes were produced by refolding inclusion bodies in the following amounts: 30 mg of α‐chain, 10 mg of β2‐microglobulin and 5 mg of peptide (GenScript) into 200 mL of buffer (100 mm Tris–HCl pH 8.0, 400 mm l ‐arginine, 500 μm glutathione oxidized, 5 mm glutathione reduced and 20 mm EDTA [ethylenediaminetetraacetic acid]) (Sigma Aldrich, Sydney, Australia). The refold mixture was dialyzed into 10 mm Tris–HCl pH 8.0 and the pHLA complexes were purified using anion exchange chromatography (HiTrap Q, Cytiva, Marlborough, MA, USA).
26 (link) In brief, DNA plasmids (GenScript, Hong Kong, China) encoding the HLA‐B*35:01, HLA‐B*35:03 and HLA‐B*35:05 heavy chain (1–275 amino acid) and β2‐microglobulin were transformed into the BL21 strain of Escherichia coli cells. Both proteins were expressed separately as inclusion bodies and purified from the transformed E. coli cells. Soluble pHLA complexes were produced by refolding inclusion bodies in the following amounts: 30 mg of α‐chain, 10 mg of β2‐microglobulin and 5 mg of peptide (GenScript) into 200 mL of buffer (100 m
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!