Paul.xu: Created page with "Transform E.coli strain BL21 (DE3) with cloned E.coli MutS (e.g. pET30b) .
Induction.
Spin down cells.
Resuspend in lysis buffer (20 mM Tris–HCl, pH 8.0, 0..."

2014-09-07T14:32:58Z

Created page with "Transform E.coli strain BL21 (DE3) with cloned E.coli MutS (e.g. pET30b) . Induction. Spin down cells. Resuspend in lysis buffer (20 mM Tris–HCl, pH 8.0, 0..."

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Transform E.coli strain BL21 (DE3) with cloned E.coli MutS (e.g. pET30b) . 

Induction. 

Spin down cells. 

Resuspend in  lysis buffer (20 mM Tris–HCl, pH 8.0, 0.5 mM 2-mercaptoethanol, 1 mg/ml lysozyme and 250 mM PMSF) 

lyse by sonication. 

Load to 5~10 ml Hi-trap Ni-column pre-equilibrated with binding buffer (20 mM Tris–HCl, pH 8.0, 0.5 mM 2-mercaptoethanol and 500 mM NaCl). 

Wash the column with 5 mM imidazole

Elute with 300 mM imidazole.

Load to Q HP or MonoQ column equlibrated with buffer  (20 mM Tris–HCl, pH 8.0, 1 mM EDTA and 1 mM DTT).

Elute with linear gradient 

[[Category:Protocols]]

Purification of Ecoli-Muts - Revision history

Paul.xu: Created page with "Transform E.coli strain BL21 (DE3) with cloned E.coli MutS (e.g. pET30b) . Induction. Spin down cells. Resuspend in lysis buffer (20 mM Tris–HCl, pH 8.0, 0..."

Paul.xu: Created page with "Transform E.coli strain BL21 (DE3) with cloned E.coli MutS (e.g. pET30b) .
Induction.
Spin down cells.
Resuspend in lysis buffer (20 mM Tris–HCl, pH 8.0, 0..."