MutL purification

Contents

1. Transformation
2. Overexpression
3. Ni-column
4. Heparin column
5. Superex 200

1. Transformation

1. Transform mutL plasmid into BL21(DE3), o/n at 37C. Store at Room temperature until use.
2. In the morning, put a colony into 5mL LB/carb (4x) at 37C for 6h.
3. In the evening, put the culture in 60mL LB/carb (4x) at 37C overnight.
4. Also prepare autoclaved 1L LB (4x).

2. Overexpression

1. Inoculate 10mL of preculture into 1L LB/carb at 37C until OD600 reaches ~ 0.5.
2. Change temperature to 20C and induce with 1mM IPTG for 4h.
3. Harvest cells at 4000rpm for 15 min.
4. Resuspend pellet in 100mL lysis buffer for all 4L culture.
5. Divide into 3 50mL conical tubes. You can freeze in liquid N2 and store in -80C for later use.

Buffers for lysis:

• 20mM Tris-HCl (pH 8.0)
• 0.1M NaCl
• 1mM betamercaptoethanol (BME)

3. Ni-column

1. Prepare thawed cultures (~40mL).
2. Sonicate to lyse cells. (75% output, 10s on, 50s off, total 2min on ice)
3. Centrifuge at 30k rpm for 30min.
4. Wash the Ni-column with 10x cv water.
5. Equilibrate with buffer A/B(6% of buffer B = 30mM).
6. Use 50mL superloop to load sample. (use 1mL/min flow rate)
7. Wash sample with 50mL of 6% buffer B.
8. Elute with gradient from 30mM - 500mM Imidazole for 120mL. Use flow rate 3mL/min for 40 min.
9. Check the fractions of peak with 10% or 12% SDS-PAGE.

Buffer B for Ni-column:

• 20mM Tris-HCl (pH 8.0)
• 0.1M NaCl
• 10% glycerol
• 500mM imidazole
• 1mM betamercaptoethanol (BME)
• Buffer A is same as buffer B except for no imidazole added

4. Heparin column

1. Load elution fractions containing MutL. Use 0.8mL/min flow rate.
2. Elute with 10% - 100% gradient of NaCl. (100mM - 1M NaCl) 18mL at 0.8mL/min flow rate.
3. Check the fractions on SDS-PAGE.

Buffer B for heparin column:

• 20mM Tris-HCl (pH 8.0)
• 1M NaCl
• 10% glycerol
• 1mM betamercaptoethanol (BME)
• Buffer A is same as buffer B except for no NaCl added

5. Superex 200

1. Concentrate fractions from previous column (~1mL).
2. Run through Superdex 200 16/600. Flow rate can be 1mL/min.

Buffers for Superdex 200:

• 20mM Tris-HCl (pH 8.0)
• 0.5M NaCl
• 10% glycerol
• 1mM betamercaptoethanol (BME)