Team:University of Chicago/Notebook/DH5-alpha competent cells

TB Solution
55mM MnCl2. Adjust to final volume. Sterilize by filtration with 0.45um filter and store at 4°C
 * 10mM PIPES
 * 15mM CaCl2
 * 250mM KCl
 * Dissolve in nanopure water and adjust pH 6.7 with KOH or HCL (solutes will dissolve as you do this) and then add

LB Media
(per 1L batch)
 * 30 g LB mix
 * Dissolve in ddH2O, filling to 1L.
 * for 20mM MgSO4 FIRST add 16mL 1M MgSO4 solution THEN fill to 1L

Procedure
overnight culture. Grow at room temp (23°C) with good aeration (250rpm) to an A600 of 0.4-0.6. freeze in dry ice/EtOH bath. Store -70°C. Typically, competency about 5X 106 cfu/ug DNA. Note, improves after freezing. Cells good for a year and counting.
 * 1) Inoculate a 3ml overnight of E.coli in LB+20 mM MgSO4.
 * 2) Next morning, inoculate 250 ml LB+20 mM Mg++ in a 2L flask with about 2ml
 * Note: TEMPERATURE IS IMPORTANT! At 37C cells will grow up to proper OD in ~3 hours. A faster growing time, however, compromises efficiency, so choose temperature accordingly.
 * 1) Place cells 10 min on ice. Transfer to a sterile bottle and spin 3K, 10', 4°C.
 * 2) Resuspend pellet in 80 ml cold TB (swirl cells in bottle). Leave 10’/ice.
 * 3) Spin cells 3K, 10', 4°C.
 * 4) Resuspend cells in 20 ml cold TB then add 1.5 ml DMSO. Leave 10'/ice.
 * 5) Dispense into 220 ul and 525 ul aliquots (in cold sterile tubes) and