Chemical Transformation of E. coli

Purpose

To introduce DNA into E. coli cells

Theory

E. coli transformation is an important step that allows the introduction of heterologous DNA using plasmid vectors or introducing mutations via homologous recombination events.

Equipment

• 4°C Centrifuge

• 42°C Water Bath

• 37°C Shaking Incubator

Materials

• Bacto Agar

• Bacto Tryptone

• Bacto Yeast Extract

• Selective Agar Plates

• Calcium Chloride

• Magnesium Chloride

• Magnesium Sulfate

• Manganese Chloride

• MOPS

• Potassium Acetate

• Potassium Chloride

• Rubidium Chloride

• Sodium Chloride

• Glucose

• Glycerol

• Acetic Acid

• Sodium Hydroxide

• Potassium Hydroxide

• 50 ml sterile polypropylene centrifuge

• tubes (i.e Corning 430829)

• 2 ml sterile, screw cap, conical bottom

• tubes (i.e. Phenix SCS-02S)

• 5–10 ml glass vials or autoclaveable

• screw cap tubes

Solutions & buffers	Step 1 Psi Media
Component	Amount/liter
Tryptone	20 g
Yeast Extract	5 g
MgCl2	5 g
Adjust to pH 7.6 with KOH Add water to 1 liter and autoclave

Tip You can also use LB medium (low salt) supplemented with 4 mM MgSO4 and 10 mM KCl or SOB (also commercially available)

Tfb I (Transformation Buffer I)
Component	Final concentration	Stock	Amount/400 ml
Potassium acetate	30 mM	-	1.18 g
RbCl2	100 mM	-	4.84 g
CaCl2 -2H2O	10 mM	-	0.59 g
MnCl2	50 mM	-	3.96 g
glycerol	15% v/v	-	60 ml
pH 5.8 with dilute acetic acid. Add water to 400 ml and filter sterilize

Tfb II (Transformation Buffer II)
Component	Final concentration	Stock	Amount/100 ml
MOPS	10 mM	-	0.21 g
CaCl2-2H2O	75 mM	-	1.1 g
RbCl2	10 mM	-	0.12 g
Glycerol	15% v/v	-	15 mls
pH 6.5 with dilute NaOH. Add water to 100 ml and filter sterilize

Step 2
LB Agar (Miller’s high salt)
Component	Amount/liter
Tryptone	10 g
Yeast Extract	5 g
NaCl	10 g
Agar	15 g
Adjust to pH 7.2 (~0.2 ml of 5 N NaOH) Add water to 1 liter and autoclave, add appropriate antibiotic when cool and pour plates

Tip LB Agar is available in premixed commercial preps

SOC media
Component	Final concentration	Stock	Amount/100 mls
Yeast extract	0.5%	-	0.5 g
Tryptone	2%	-	2.0 g
NaCl	10 mM	3 M	0.33 ml
KCl	2.5 mM	1 M	0.25 ml
MgCl2	10 mM	1 M	1 ml
MgSO4	10 mM	1 M	1 ml
Glucose	20 mM	1.1 M	1.82 ml
Add water to 100 mls aliquot in 5–10 ml samples and autoclave

Protocol

Duration
	Preparation	about 15 minutes
	Protocol	about 4–6 hours
Preparation	Make sure you have selective agar plates on hand.
	Pick a single colony from a freshly streaked plate and inoculate a small culture (2–5 mls). Grow overnight at 37°C.
	TfbI and TfbII should be stored at 4°C to make sure they are chilled

Step 1	Prepare competent cells
Overview Duration	Grow cells to mid-log and make competent by chemical treatment 3–5 hours
1.1	Inoculate 100 mls of Psi broth with 0.5 ml of overnight culture and incubate at 37°C with vigorous shaking.
1.2	When A600 reaches 0.4–0.5 place on ice and chill 5–10 minutes
1.3	Transfer cells to 50 ml sterile chilled polypropylene centrifuge tubes. Pellet cells at 4°C for 5 minutes at 5,000 × g
Tip	If using other tubes they must be very clean and free of soap residue. Cells and transformation buffers should be kept cold at all times. It is also preferable to use chilled pipets and do everything in the cold room if possible.
1.4	Discard supernatant carefully and gently resuspend cell pellet in 0.4 volume ice cold TfbI (20 mls for each 50 ml tube). Do not vortex and keep on ice while resuspending.
1.5	Incubate cells on ice for 15 minutes.
Tip	Some protocols incubate for only 5 minutes and cells can be left on ice for longer periods (i.e. 1–2 hrs) without any harm
1.6	Pellet cells at 4°C for 10 minutes at 2,000 × g
1.7	Discard supernatant carefully and gently resuspend in 0.02 volume (1 ml for 50 mls of culture) TfbII while keeping on ice.
1.8	Aliquot 50 microliters into 2 ml sterile screw cap tubes with conical bottom and no skirt. Do not use standard 1.5 ml conical microfuge tubes – they don’t work well in the heat shock step of the transformation.
1.9	Flash freeze in dry ice ethanol bath or liquid nitrogen and store at −80°C
Step 2	Transform competent cells
Overview Duration	Introduce DNA into competent cells 2 hours
2.1	Equilibrate a water bath to 42°C. A dry block will work if the tube fits snugly, but is not as good as the water bath.
2.2	Thaw 1 vial of competent cells on ice for each transformation. Handle gently since cells are sensitive to temperature changes and mechanical lysis.
2.3	Add 1 to 5 microliters of DNA (10 pg to 100 ng) to a vial of thawed competent cells. DO NOT VORTEX OR PIPETT UP AND DOWN. Supercoiled DNA is transformed more efficiently than ligated DNA.
2.4	Incubate on ice for 30 minutes
2.5	Heat shock cells for 30 seconds at 42°C. Do not go any longer or shake the cells
2.6	Remove from the water bath and place on ice for 2 minutes
2.7	Add 250 microliters of SOC media to each vial
2.8	Make sure cap is tight and incubate tube on its side in a 37°C shaking incubator (200–250 rpm) for 1 hour.
2.9	Spread from 20 – 200 microliters on an appropriate selective plate. The plates should be at room temperature or prewarmed to 37°C. Incubate overnight at 37°C.
Tip	Transformed cells can be stored at 4° C for 24–48 hours with minimal loss of viability. Transformation efficiency varies depending on DNA.