Klymkowsky Lab On-line Methods
Making competant bacteria

Table of Methods 3 December 2002

from Ausubel et al 1985


Typically we use DH5a cells since they are tolerant to the source of the DNA
(B or K strain).

The same protocol will work with BL21 cells (for protein synthesis)
and other bacterial strains.

  1. streak out the cells from a -80°C stock on an LB plate -- do not use an LB-AMP plate; the bacteria do not have plasmids in them they will not grow in the presence of AMP.
  2. Inoculate a single colony of E. coli cells into 50 ml LB media, grow overnight at 37°C with moderate shaking (250 rpm)
  3. Inoculate 4 ml of the culture into 400ml LB media in a 2 L sterile flask
    grow at 37°C with shaking until OD590 is ~0.375
  4. Aliquot into ten 40ml sterile polypropylene tubes and leave on ice for 10 min.
  5. Centrifuge cells for 7min at 3000rpm, 4°C, in Sorvall rotor
  6. Discard the supernatant; resuspend each pellet in 8 ml ice-cold calcium chloride solution. Resuspend cells by gentle swirling!
  7. Centrifuge cells for 5 minutes at 2000 rpm at 4°C -- Discard supernatant
    Resuspend cells as before with calcium chloride solution (8ml per tube).
  8. Centrifuge cells for 5 minutes at 2000 rpm at 4°C -- Discard supernatant
  9. Resuspend by swirling gently in 1.8 ml of ice cold calcium chloride solution
    Pellets must be resuspended completely.
  10. Dispense 0.1 mL cells into prechilled, sterile 1.5ml microfuge tubes and freeze at -80°C.

Calcium chloride solution:

60mM CaCl2
15% glycerol
10mM PIPES, pH 7.0

Recipe for LB media:

5 g NaCl
10 g casamino acids or tryptone
5 g yeast extract
1ml 10M NaOH pour
water to 1 litre - use distilled water


LB amp plates:

add 15g agar (sigma A7002) and autoclave

add 1ml 100 mg/ml ampicillin (AMP) per litre after liquid has cooled to below 60°C

pour plates 0.5 cm thick

Table of Methods