Jhdavis::ClpXPrep: Difference between revisions
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(New page: ==Express from BLR (pACYC-derived plasmid) at RT for 3 hours post-induction (induce at OD = 0.8 after cooling to RT for 25 mins) ==Lyse in 20 mM Hepes, 400 mM NaCl, 100 mM KCl, 20 mM Imida...) |
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#Express from BLR (pACYC-derived plasmid) at RT for 3 hours post-induction (induce at OD = 0.8 after cooling to RT for 25 mins) | |||
#Lyse in 20 mM Hepes, 400 mM NaCl, 100 mM KCl, 20 mM Imidazole, 10% glycerol pH 7.6 | |||
#Lysozyme + french press with PMSF (generates foam in french press | |||
#Spin down, add sup to 4 mL Ni-slurry | |||
#2 washes with lysis buffer+PMSF on bench, add to column | |||
#3x washes in column, with 1 wash used to resuspend the column | |||
#0.5 mL elutions with lysis buffer - PMSF + 250 mM Imidazole | |||
#pool fractions by bradford | |||
#S300 in 50 mM Tris, 300 mM KCl, 0.1 mM EDTA, 1 mM DTT, 10%glycerol pH 7.5 | |||
#Check by gel | |||
Latest revision as of 08:15, 19 June 2008
- Express from BLR (pACYC-derived plasmid) at RT for 3 hours post-induction (induce at OD = 0.8 after cooling to RT for 25 mins)
- Lyse in 20 mM Hepes, 400 mM NaCl, 100 mM KCl, 20 mM Imidazole, 10% glycerol pH 7.6
- Lysozyme + french press with PMSF (generates foam in french press
- Spin down, add sup to 4 mL Ni-slurry
- 2 washes with lysis buffer+PMSF on bench, add to column
- 3x washes in column, with 1 wash used to resuspend the column
- 0.5 mL elutions with lysis buffer - PMSF + 250 mM Imidazole
- pool fractions by bradford
- S300 in 50 mM Tris, 300 mM KCl, 0.1 mM EDTA, 1 mM DTT, 10%glycerol pH 7.5
- Check by gel
