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Making Ultra-competent E.coli (DH5-alpha) - the Inouye Method

   
This "simple and efficient method" is the result of painstaking optimization of bacterial growth and transformation conditions by Inouye et al. The bacteria take a while to grow (1-2 days), but the actual preparation time is less than two hours, and you only have to heat-shock for 30 seconds.
 
Reference: Inouye et al (1990). High efficiency transformation of Escherichia coli with plasmids. Gene 96:23-28.
 

PROCEDURE

  • Inoculate 250 ml SOB media with 25 ul of an overnight culture (grown at 37OC in LB).
  • Grow, with shaking, at 18OC until OD600 = 0.6 to 0.75 (about 48 hours for 18OC; 24 hours at 20OC).
  • Place culture on ice for 10 minutes.
  • Pellet bacteria 10 min, 2500 x g in a refrigerated centrifuge (refer to this RPM to RCF calculator).
  • Gently resuspend cells in 80 ml ice-cold transformation buffer (TB).
  • Ice for 10 minutes.
  • Pellet bacteria for 10 min, 2500 x g in a refrigerated centrifuge.
  • Gently resuspend cells in 20 ml ice-cold TB.
  • Add 1.5 ml DMSO (final concentration = 7%)
  • Ice for 10 minutes
  • Aliquot competent cells and freeze at -80OC (or in liquid nitrogen for long-term storage).

TRANSFORMATION

  • Thaw cells on ice.
  • Pre-dispense DNA solution (up to 20 ul) or ligation mixture (1-5 ul optimum) into polypropylene tubes. Place on ice.
  • Add 50-200 ul of thawed competent cells; swirl gently to mix.
  • Incubate on ice for 30 minutes.
  • Heat-shock in a 42OC water bath for 30 seconds, with slight agitation (swirl tubes gently).
  • Ice briefly; add 0.8 ml SOB (LB may be used, with a slight decrease in transformation efficiency).
  • Incubate, with shaking, at 37OC for up to one hour.
  • make serial dilutions in LB media (e.g. 101, 102, 103, 104, and so on).
  • spread 100 ul of each dilution onto LB-agar plates containing selective antibiotic (see recommended antibiotic concentrations). I recommend using glass beads to spread bacteria.
  • Transformation efficiencies of up to 1 x 109 colony-forming units (cfu)/ug DNA can be obtained!

NOTES

  • Bacterial growth temperature: very important factor in making competent cells. Growing bacteria between 18OC - 25OC gives good competence, with 18oC yielding slightly higher transformation efficiency.
  • Cell density: when growing cells at 37OC, transformation efficiency peaks sharply at OD600 = 0.45, and drops precipitously at slightly higher ODs. Lower temperatures (18OC-25OC) allow a broad window of optimal cell density: OD600 anywhere between 0.25 to 1.0 will give higher transformation efficiency than cells grown to optimal density at 37OC.
  • Volume of DNA: the volume of DNA added, up to 20 ul, has little influence on transformation efficiency.
  • Polypropylene tubes: Use polypropylene tubes. Glass decreases transformation efficiency, and polystyrene supposedly gets dissolved by DMSO (which you probably want to avoid).
  • Heat shock time: duration of heat shock makes a much bigger difference than the temperature; 30 seconds (with agitation) is optimal, and anywhere from 37-52OC.
  • Freezing: freezing before use increases transformation efficiency, and decreases only gradually even when stored at -80OC for weeks or even months.
Antibiotic
 recommended working concentration (ug/mL)
ampicilllin 
 100
carbencillin 
 100
chloramphenicol 
 33
kanamycin 
 30
streptomycin 
 25
tetracycline 
 15
zeocin 
 25
SOB medium Ingredients [final] for 500 mL for 1 L
  tryptone [2%] 10 g 20 g
  yeast extract [0.5%] 2.5 g 5 g
  NaCl [10 mM] 0.25 g or 5 ml of 1M stock 0.5 g or 10 ml of
  KCl [2.5 mM] 1.25 ml of 1M stock 2.5 ml of 1M stock
  MgCl2 [10 mM] 5 ml of 1M stock (sterile) 10 ml of 1M stock (sterile)
  MgSO4 [10 mM] 5 ml of 1M stock (sterile) 10 ml of 1M stock (sterile)
  pH 6.7-7.0    
  • Dissolve tryptone, yeast extract, NaCl, and KCl in dH2O (90% of final volume).
  • Autoclave*
  • Add MgCl2 and MgSO4
  • Store at room temperature.
*I'm not sure why you have to autoclave this before adding MgCl2 and MgSO4 - it works fine if you just mix everything and autoclave at the end.
Transformation Buffer (TB) ingredients [final] for 500 mL for 1 L
  pipes [10 mM] 1.512 g 3.024 g
  CaCl2 [15 mM] 1.1025 g 2.205 g
  KCl [250 mM] 9.32 g 18.64 g
  MnCl2 [55 mM] 5.44 g 10.88 g
  • Dissolve all ingredients in dH2O and adjust to final volume. Sterilize by 0.2 micron filtration. Best not to autoclave - I did once and something formed a yucky brown precipitate. Store at 4OC.

Last updated 4 October 2007
millions and millions

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