Paul.xu at 19:12, 16 May 2014

2014-05-16T19:12:24Z

New page

== For His-Tag fusion of Taq-Muts ==

1. Induce with 0.2 mM IPTG o/n at 37 oc. 2. Lyse cells with lysis buffer 25 mM Tris-HCl, 150 mM NaCl, 0.1 mM dithiothreitol, 0.1 mM EDTA. 3. Heat lysate at 70 °C for 15 min 4. Spin at 25000 rpm for 30 min 5. Load supernatant to a 15 ml Ni column, wash with Buffer A containing 5 mM Imidazole. 6. Elute with Buffer B: 25mM Tris-HCl, pH 8.0, 250 mM Imidozole, 100 mM NaCl, 5% (v/v) glycerol, 0.5 mM beta-ME. 7. Dilute with 1 vol of dilution buffer: 25mM Tris-HCl, pH 8.0, 5% (v/v) glycerol, 0.5 mM beta-ME. 8. Load to a QFF column. 9. Wash with dilution buffer 25mM Tris-HCl, pH 8.0, 5% (v/v) glycerol, 0.5 mM beta-ME. 10. Run linear gradient of 100 mM to 1 M NaCl in 25 mM Tris-HCl, pH 8.0, 5% (v/v) glycerol, 0.5 mM beta-ME on FPLC 11. MutS protein should be eluted at approximately 260 mM NaCl. 12. Concentrate and then dialyse 2 times with dialysis buffer containing 1 mM DTT and 50% glycerol. 13. measure protein concentration, Make 0.5 mg aliquotes 14. Store protein at -80 °C.

Yield should be at least 2 mg per L culture. 

[[Category:Protocols]]

Purification of Taq Muts - Revision history

Paul.xu at 19:12, 16 May 2014