Purification of Human DNA Polymerase μ

Truncated human pol μ (loop 2 deletion mutant, hPol μ Δ2) was overexpressed in BL21-CodonPlus(DE3)-RIL cells (Invitrogen) at 16 °C overnight and purified as previously described^{25 (link)}. Cells were lysed by sonication in 25 mM Tris/HCl, pH 8.0 (25 °C), 500 mM NaCl, 5% glycerol, 1 mM DTT. Pol μ was batch purified on Glutathione Sepharose CL-4B resin (GE Healthcare) and eluted by TEV cleavage overnight at 4 °C. Size-exclusion chromatography was performed on a Superdex 200 26/600 column in 25 mM Tris/HCl, pH 8.0 (25 °C), 100 mM NaCl, 5% glycerol, 1 mM DTT, 1 mM EDTA. Pol μ was then dialyzed into 25 mM Tris/HCl, pH 8.0 (25 °C), 100 mM NaCl, 5% glycerol, 1 mM DTT, concentrated to 11 mg ml⁻¹, and stored at −80 °C. Expression plasmids were sequenced in both directions to confirm the expected sequence by Genewiz, Inc.