---+ Preparing Chemically Competent Cells using the Inoue Method Adapted from _Molecular Cloning: A Laboratory Manual 3rd Ed._, Sambrook and Russell (2001) ---++ SUPPLIES: ---+++ Equipment: * Floor Centrifuge * Sterile 500mL Centrifuge Bottles * Shaking Incubator or Shaking Platform and 1L flask clamps * 42°C Heating Bath or Block * Sterile 1L Flasks ---+++ Consumables: * Liquid Nitrogen * Polypropylene Microcentrifuge Tubes ---+++ Buffers and Solutions: * [[ProtocolsRecipesSOB][SOB Medium (20mM MgSO4)]] * [[ProtocolsRecipesSOB][SOC Medium]] * 0.5 M PIPES (pH 6.7): * Dissolve 15.1g PIPES in 80mL MilliQ / UltraPure H2O, adjust pH to 6.7 w/ 5M KOH (measure 1mL aliquots in a 15mL falcon tube to avoid contaminating stock solution). Adjust volume to 100mL and filter sterilize, divide into 10mL aliquots in 15mL Falcon tubes, and store at -20°C * Transformation Buffer: | *Reagent* | *Amount / L* | *Final Conc.* | | MnCl<sub>2</sub> • 4H<sub>2</sub>O | 10.88 g | 55 mM | | CaCl<sub>2</sub> • 2H<sub>2</sub>O | 2.20 g | 15 mM | | KCl | 18.65g | 250 mM | | PIPES (0.5 M, pH 6.7) | 20 mL | 10 mM | | H<sub>2</sub>O | to 1 L | | * * Filter sterilize and store 40mL aliquots in 50mL Falcon Tubes at -20°C ---++ PROTOCOL 1 ) First thing in the morning, seed a starter culture in 25mL LB in a 250mL flask with a single colony from a freshly grown overnight plate. Grow this culture 6-8 hours @ 37°C, 300 rpm. * Alternatively, you can prepare frozen starter cultures by growing a 50mL culture overnight, dividing into aliquots, adding 0.25 volumes 80% Glycerol, and freezing at -80°C 1 ) At the end of the day (~6pm) inoculate 3 1L flasks containing 250mL SOB with 10mL, 4mL, and 2mL of fresh or frozen starter culture. Incubate these on a shaking platform at room temp (~18°-22°C), shaking at 200-250 rpm. * The point of this is to make sure at least one of these cultures will reach the right OD when you come in the next morning. 1 ) The following morning, read the OD<sub>600</sub> of all three cultures. Measure every 45min until one of the cultures reaches an OD<sub>600</sub> of 0.55; keep this culture and discard the other two. 1 ) Place the culture flask in an ice bath for ~10min. 1 ) Harvest cells by centrifuging at 2500xg for 10min at 4°C 1 ) Pour off supernatant and invert the centrifuge bottle on a paper towel for two min; remove any remaining drops of medium with a pipette 1 ) Resuspend cells by gently swirling in 0.32x the original culture volume ice-cold transformation buffer (e.g. 80mL buffer if you started w/ a 250mL culture) 1 ) Harvest cells by centrifuging at 2500xg for 10min at 4°C 1 ) Pour off remaining medium and remove any remaining drops 1 ) Resuspend cells in 0.08x original culture volume ice-cold transformation buffer (e.g. 20mL buffer if you started w/ a 250mL culture) 1 ) Add DMSO to a final concentration of 7% (so add 1.5mL if your resuspended cell solution is 20mL) 1 ) Dispense 50uL aliquots into pre-chilled centrifuge tubes, then close tubes tightly and snap-freeze in liquid nitrogen 1 ) Store cells at -80°C until needed -- <span style="background-color: transparent">Main.ColinBrown</span><span style="background-color: transparent"> - 15 Dec 2016</span>
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Topic revision: r2 - 2017-02-23 - ColinBrown