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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Revision as of 08:14, 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
