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. 2014 Feb 5;9(2):e82624. doi: 10.1371/journal.pone.0082624

Figure 1. Schematic diagram of the DNA decontamination of Phi29 DNA polymerase.

Figure 1

GST-fused Phi29 DNA polymerase (GST-ø29pol) is expressed in E. coli and affinity purified by glutathione sepharose 4B resin (GS4B) of the cell lysate after polyethylenimine (PEI) precipitation to remove the host DNA. Then, GST-ø29pol is treated with ethidium monoazide (EMA) and irradiated with visible light (VL) to reduce both ssDNA and dsDNA, which may be captured in GST-ø29pol. After washing away the free EMA, the remaining oligonucleotides are reduced by the endogenous 3′-5′ exonuclease activity of the polymerase. Finally, DNA-free Phi29 DNA polymerase is separated from GS4B by digestion with PreScission protease and collected.