This protocol is a larger scale variant of one due to Mike Scott, Department of Neurology, UCSF. Streak out the desired strain on an LB plate. The next day, innoculate a single colony into 20ml TYM broth (recipes below) in a 250ml flask. Grow the cells to midlog phase (OD$_{600}$ $\approx$0.2-0.8), pour into 2l flask containing 100ml TYM, continue vigorous agitation until cells grow to 0.5-0.9 OD, then dilute again to 500ml in the same vessel. When cells grow to OD$_{600}$ 0.6, put flask in ice-water, and shake gently to assure rapid cooling. When culture is cool, spin 4.2k rpm, 15 minutes (J6). Pour off supernatant, resuspend pellet in $\approx100$ml cold TfB I (below) by gentle shaking on ice. Respin in same bottle 4.2krpm, 8 minutes (J6). Pour off supernatant, resuspend pellet in 20ml cold TfB II by gentle shaking on ice. Aliquot 0.1 to 0.5 ml aliquots in prechilled microfuge tubes, freeze in liquid nitrogen, and store at $-70$\degrees. For transformation, remove an aliquot, thaw at room temperature until just melting, place on ice, add DNA, let sit on ice 15-30 minutes, incubate at 37\degrees\ for 5 minutes (6 minutes for 0.5ml aliquots), dilute 1:10 in LB and grow for 90 minutes before plating or applying antibiotic selection. Alternatively, the heat-pulsed transformation mix can be plated directly on antibiotic plates onto which a thin (4-5ml) layer of antibiotic-free LB agar has been poured just before plating. {\bf Media and Buffers:} TYM: 2\% Bacto-Tryptone, 0.5\% Yeast Extract, 0.1M NaCl, 10mM MgSO$_4$ (can be added before autoclaving). TfB I: 30mM KOAc, 50mM MnCl$_2$, 100mM KCl, 10mM CaCl$_2$, 15\% (v/v) glycerol. TfB II: 10mM Na-MOPS, pH 7.0, 75 mM CaCl$_2$, 10mM KCl, 15\% glycerol.