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{{:Team:EPF-Lausanne/Templates/ProtocolHeader|title=Competent cells preparation (by Inoue)}} ==Materials== * Plate of cells streaked for single colonies * [[SOB]] * Ice * [[TB buffer]] * DMSO * Dry Ice (or liquid nitrogen) ==Glassware & equipment== * 2 liter erlenmeyer flask (no detergent residue, rinse with 70% ethanol and DI water) * 220 ml conical centrifuge tubes BD 35 2075 * Eppendorf 5410R refrigerated centrifuge with conical adapters ==Preparation== # Pick a 10 - 12 large single colonies (2-3 mm dia) from your source plate and inoculate 500 ml of sterile SOB medium (do not use LB) in a 2 liter flask. Save some medium as an OD blank. # Grow to an OD of 0.6 with vigorous shaking (200-250 rpm) at 18 degrees (important). This is slow -- approximately 35-40 hours. # Prechill the centrifuge to 4 degrees # Remove from the incubator and place on ice for 10 minutes # Transfer to two 220 ml centrifuge tubes and spin at 3220 x g for 10 minutes at 4 degrees # Drain the medium and resuspend each pellet first in 5 ml of ice cold TB. Add an additional 75 ml of cold TB buffer and resuspend. # Place on ice for 10 minutes # Spin down as above. # While spinning, add 1.4 ml of DMSO to 18.6 ml of TB (7% DMSO mixture) # Resuspend each pellet in 20 ml of cold TB-DMSO mixture # Incubate on ice for 10 minutes # Dispense cells into pre-chilled tubes # Freeze cells in a dry ice / ethanol bath and store at -80 degrees indefinitely ==Thoughts on improvements== * "Methods in Yeast Genetics" book (Amberg05) suggests growth the SOB + 300 mM NaCl * They also control pH at 7.5, which may be a major issue * Centrifuging in flat bottom centrifuge tubes may make pellet resuspension easier and less damaging * Length of time on ice prior to transformation may make a big difference * The Hanahan protocol specifies dry pure DMSO, while Inoue says it doesn't make a difference. Let's see. * Warm plates for growing cells after transformation are claimed to be 2x to 4x more efficient. {{:Team:EPF-Lausanne/Templates/Footer}}

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