---+ 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 ---++ PREPARING CELLS 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 2.) 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 com in the next morning. 3.) 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. 4.) Place the culture flask in an ice bath for ~10min ---++ STEP 2: IMAC PURIFICATION W/ GRAVITY COLUMN -- Main.ColinBrown - 15 Dec 2016
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Topic revision: r1 - 2016-12-15 - ColinBrown