Making Competent DH5a bacteria

TB: 10mM PIPES

15mM CaCl₂

250mM KCl

make to 800ml in dH₂O

pH to 6.7 with KOH (all fresh)

Add MnCl₂ to 55mM

Filter sterilize

Store at 3^o C

Streak out cells on LB agar plate

Grow ON at 37^o C

Innoculate 250 ml SOB in a 2 Liter flask with 10-12 large colonies (~0.5 cm of a streak of bacteria) X 3=750ml cells

Shake at RT to OD 600 = 0.4-0.6

When cells reach OD, chill on ice at least 10 minutes

Have TB on ice

Have 500ml sterile centrifuge bottles at 4^o C

Put 250ml cells into each 500ml bottle

Spin 3K for 10 minutes at 4^o C

R/S each pellet in 80ml cold TB (=240ml TB)

Transfer to 6 50ml conical tubes

Icw 10 minutes

Spin 2L for 10 minutes at 4^o C in TC room table top centrifuge

R/S each tube in 10ml TB (=60ml TB)

Pool

Add DMSO to final 0.7% (=.42ml total)

Ice 10 minutes

Aliquot 100-200ul/1.7ml tube

Snap freeze in liquid nitrogen

Store at -80^o C