Codon-optimized SUDV MTase+CTD synthetic genes (Biomers) were cloned into a pET14b vector for expression in bacteria. Mutations were introduced by PCR-amplifying the wild-type sequence using primers carrying the mutations, using the Turbo DNase (Ambion). PCR-products were purified using the Wizard SV PCR Clean-Up System (Promega). Transformed Escherichia coli T7 bacteria (NEB) were cultured at 30°C until an O.D. of 0.6 was reached, after which the temperature was changed to 17°C and IPTG (Sigma) was added to 20 μM. The next day, bacteria were spun down (8000 × g for 10 min at 4°C) using a Sorval Lynx 6000 centrifuge (Thermo), and pellets were stored at –80°C.
Sorval lynx 6000 centrifuge
Manufactured by Thermo Fisher Scientific
The Sorval Lynx 6000 centrifuge is a high-performance laboratory equipment designed for a variety of applications. It features a powerful motor and can achieve high-speed centrifugation up to 22,000 rpm, with a maximum RCF of 50,800 x g. The Sorval Lynx 6000 is suitable for a range of sample types and volumes.
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2 protocols using sorval lynx 6000 centrifuge
1
Codon-optimized SUDV MTase+CTD expression
2
Recombinant RSV MTase-CTD Expression
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The codon-optimized RSV MTase-CTD synthetic gene (Genscript) was cloned in a modified pGEX-4T-3 vector with an N-terminal 6x His-tag for bacterial expression. Mutants were generated by PCR amplification using the Pfu DNA polymerase kit (Promega) and primers carrying the mutations (S2 Table ). Proteins were produced in Escherichia coli chaperone competent pGro7/BL21 cells (Takara). Cells were grown in Luria broth with L-arabinose (1 g/L) at 30°C until the OD600 value reached 0.6. Protein expression was induced with 20 μM IPTG at 16°C, overnight. Bacteria were then spun down (8000 rpm, 4°C, 10 min) using a Sorval Lynx 6000 centrifuge (Thermo Scientific), and pellets were stored at -80°C.
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