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Finkelstein Lab Wiki

MutL purification

Contents

Section titled “Contents”
  1. Transformation
  2. Overexpression
  3. Ni-column
  4. Heparin column
  5. Superdex 200

1. Transformation

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

2. Overexpression

Section titled “2. Overexpression”
  1. Inoculate 10mL of preculture into 1L LB/carb at 37°C until OD600 reaches ~ 0.5.
  2. Change temperature to 20°C 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 -80°C for later use.

Buffers for lysis:

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

3. Ni-column

Section titled “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

Section titled “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. Superdex 200

Section titled “5. Superdex 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)