Alw44I (10 U/µL)

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	To transfect linearized DNA, plasmid DNA was digested with Alw44I (Thermo Scientific) and purified with the GeneJet gel extraction kit (Thermo Scientific) prior to transfection.

Protocol tips
To transfect linearized DNA, plasmid DNA was digested with Alw44I (Thermo Scientific) and purified with the GeneJet gel extraction kit (Thermo Scientific) prior to transfection.

Publication protocol

HeLa cells were transfected with the cationic JetPei transfection reagent (Polyplustransfection). The amounts of DNA and JetPei reagent were adjusted depending on the cell culture format used, according to the manufacturer’s instructions. DNA was quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). To generate stably transfected cell lines, HeLa cells were allowed to grow and to express the drug resistance gene under nonselective conditions for 24 to 48 h after transfection. Then, cells were cultivated in standard medium supplemented with the appropriate drug for 4 to 5 weeks until outgrowth of resistant cells. Medium was changed every 2 to 3 days to avoid a loss of selection pressure. To obtain the integration rate of transfected plasmid DNA, transfections were carried out in 6-well plates. To transfect linearized DNA, plasmid DNA was digested with Alw44I (Thermo Scientific) and purified with the GeneJet gel extraction kit (Thermo Scientific) prior to transfection.

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