ProtocolsChemCompCellsInoue < Lab

---+ Preparing Chemically Competent Cells using the Inoue Method
< [[ProtocolsChemCompCellsHub][Return to Transformation Protocol]]

 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> &bull; 4H<sub>2</sub>O | 10.88 g | 55 mM |
| CaCl<sub>2</sub> &bull; 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

< [[ProtocolsChemCompCellsHub][Return to Transformation Protocol]]

-- <span style="background-color: transparent">Main.ColinBrown</span><span style="background-color: transparent"> - 15 Dec 2016</span>
Barrick Lab > ProtocolList > ProtocolsChemCompCellsInoue
Topic revision: r3 - 2020-04-09 - 15:58:32 - Main.KateElston
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