FastDigest Bst1107I

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	Ninety percent confluent cells cultured in a 25 cm2 flask were transfected with 10 μg of linearized vector pSwitch (Bst1107 I restriction endonuclease) using 25 μL of lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

Protocol tips
Ninety percent confluent cells cultured in a 25 cm2 flask were transfected with 10 μg of linearized vector pSwitch (Bst1107 I restriction endonuclease) using 25 μL of lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

Publication protocol

In this study, two different eukaryotic expression vectors were used: pBudCE4.1 (Invitrogen, Carlsbad, CA, USA) allowing for the simultaneous expression of two different genes and the GeneSwitch System (Invitrogen, Carlsbad, CA, USA) enabling induced expression in mammalian cells. For each gene in each system, two or, when needed, three different constructs were prepared: (I) native kozak sequence + gene + stop codon; (II) native kozak sequence + gene + myc/V5 epitope; (III) enhanced kozak sequence + gene + stop codon. All genes were amplified from templates with HiFi high fidelity polymerase (Kappa Biosystems, Wilmington, MA, USA) and cloned into Multi Cloning Site (MCS) of chosen vectors with the use of FastDigest restriction enzymes (Thermo Scientific, Vilnius, Lithuania). Ligation reactions were performed with the Rapid Ligation and Dephos system (Roche, Mannheim, Germany). All clones were prepared in XL10 E. coli chemically competent cells using the standard protocol [31]. All media plates were supplemented with proper antibiotics (zeocin at 50 mg/mL, ampicillin 100 mg/mL).Ninety percent confluent cells cultured in a 25 cm2 flask were transfected with 10 μg of linearized vector pSwitch (Bst1107 I restriction endonuclease) using 25 μL of lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After 24 h cells were trypsinized (Sigma-Aldrich, St. Louis, MO, USA), harvested by centrifugation (2000 rpm, 4 min) and plated at 5 × 105/well on a 6 well plate. Cells were allowed to adhere overnight and a fresh medium supplemented with appropriate concentration of hygromycin was applied. The optimal concentration of hygromycin was determined according to the manufacturer’s protocol. The medium was replaced every 3 days. After three to four weeks, separate colonies could be visible. Cells from different colonies were moved to new 96 well plates and tested by transfection with specially constructed vector pGene-EGFP and mifepristone induction. Clones that showed the same morphology as the parent strain, minimal production of EGFP without induction and high EGFP level after induction with mifepristone were assigned for further investigation.

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