WENDY CHAO'S LABORATORY PROTOCOLS

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 (the most important paper in molecular biology, IMHO):

Inouye H, Nojima H, Okayama H. High efficiency transformation of Escherichia coli with plasmids. Gene. 1990 Nov 30;96(1):23-8.

| Google Scholar | PubMed |

PROCEDURE

Inoculate 250 mL SOB media with 25 ul of an overnight culture (grown at 37°C in LB).
Grow, with shaking, at 18°C until OD₆₀₀ = 0.6 to 0.75 (about 48 hours for 18°C; 24 hours at 20°C).
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 -80°C (or in liquid nitrogen for long-term storage).

TRANSFORMATION

Thaw cells on ice.
Pre-dispense DNA solution (up to 20 microL) or ligation mixture (1-5 microL optimum) into polypropylene tubes. Place on ice.
Add 50-200 microL of thawed competent cells; swirl gently to mix.
Incubate on ice for 30 minutes.
Heat-shock in a 42°C 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 37°C for up to one hour.
Make serial dilutions in LB media (e.g. 10¹, 10², 10³, 10⁴, and so on).
Spread 100 microL 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)/microg DNA can be obtained!

NOTES

Bacterial growth temperature: This is a very important factor in making competent cells. Growing bacteria between 18°C - 25°C gives good competence, with 18^oC yielding slightly higher transformation efficiency.
Cell density: When growing cells at 37°C, transformation efficiency peaks sharply at OD₆₀₀ = 0.45, and drops precipitously at slightly higher ODs. Lower temperatures (18°C-25°C) 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 37°C.
Volume of DNA: The volume of DNA added, up to 20 microL, 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: The duration of heat shock makes a much bigger difference than the temperature; 30 seconds (with agitation) is optimal, and anywhere from 37-52°C.
Freezing: Freezing the bacteria before transforming increases transformation efficiency, and the efficiency decreases only gradually even when stored at -80°C for weeks or even months.

Antibiotic	recommended working concentration (microg/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 4°C.

Last updated 4 October 2007

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