Paul.xu: Created page with "== Purification of Taq-Muts, no tag
== 1. Grow E. coli strain such as BL21(DE3) transformed with 353 pT7A3E-HC-TaqMuts in 8L LB/AMP at 37 °C.
2. Induce for 4 h with 1 mM..."

2014-02-25T18:00:36Z

Created page with "== Purification of Taq-Muts, no tag == 1. Grow E. coli strain such as BL21(DE3) transformed with 353 pT7A3E-HC-TaqMuts in 8L LB/AMP at 37 °C. 2. Induce for 4 h with 1 mM..."

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== Purification of Taq-Muts, no tag ==

1. Grow E. coli strain such as BL21(DE3) transformed with 353 pT7A3E-HC-TaqMuts in 8L LB/AMP at 37 °C. 2. Induce for 4 h with 1 mM IPTG. 3. Spin down cells, resupend cells in 20 mM KPO4 (pH 8.0), 100 mM NaCl, 1 mM beta-ME, 0.1 mM EDTA, store cells at -20 or -80 °C. 4. Thaw cells and homogenize. 5. Heat the lysate at 70 °C for 15 min. 6. Spin at 25000 rpm for 20 min, at 4 oC. 7. Measure volume, add saturatedor solid (NH4)2SO4 to 65% saturation 8. Incubate at 4 oC for 30 min. 9. Spin for 25000 rpm for 20 min, at 4 oC. 10. Resuspend pellet in '''Buffer 1'''

 20 mM potassium phosphate, pH 8.0, 100 mM NaCl, 5% (v/v) glycerol, 1 mM beta-ME 0.1 mM EDTA

 11. Desalt the protein solution on a Sephedex G25 column using Buffer 1. 12. Load to 20 ml QFF column. 13. Wash column with Buffer 1 14. Elute with linear gradient from Buffer 1 to '''Buffer 2 '''

 20 mM potassium phosphate, pH 8.0, 1 M mM NaCl, 5% (v/v) glycerol, 1 mM beta-ME 0.1 mM EDTA

 15. MutS will be eluted at ~ 260 mM NaCl. 16. Combine fractions containing MutS. 17. Dilue protein solution with '''Buffer 3'''

 20 mM Tris-HCl or HEPES, pH 7.8, 50 mM NaCl, 5% (v/v) glycerol, 0.1 mM EDTA, 1 mM beta-ME

18. Load to a 15 ml SP FF column, collect flow through and wash. 19. Load flow through and wash to a 15 ml phoshpate cellulose column. 20. Elute protein with '''Buffer 4'''.

 50 mM Tris-HCl or 20 mM HEPES, pH 7.8, 0.4 M NaCl, 5% (v/v) glycerol, 0.1 mM EDTA, 1 mM beta-ME

 21. Load to a 10 ml hydroxyapatite column (Bio-Rad), equilibrated with Buffer 4. 22. Elute protein using a 60-ml linear gradient of 0 –120 mM potassium phosphate in HEPES buffer. MutS eluted at 60 mM potassium phosphate. 23. Combine Muts fractions and dialyse against enzyme storage buffer. 

[[Category:Protocols]]

Taq Muts - Revision history

Paul.xu: Created page with "== Purification of Taq-Muts, no tag == 1. Grow E. coli strain such as BL21(DE3) transformed with 353 pT7A3E-HC-TaqMuts in 8L LB/AMP at 37 °C.2. Induce for 4 h with 1 mM..."

Paul.xu: Created page with "== Purification of Taq-Muts, no tag
== 1. Grow E. coli strain such as BL21(DE3) transformed with 353 pT7A3E-HC-TaqMuts in 8L LB/AMP at 37 °C.
2. Induce for 4 h with 1 mM..."